Mary Sheats

Our goal for this research is to determine whether cell free DNA (cfDNA) is a useful airway biomarker for the diagnosis of neutrophilic EA. We hypothesize that cfDNA will be increased in tracheal wash (TW) and/or bronchoalveolar lavage (BAL) fluid supernatant from horses with clinical signs and diagnosis of neutrophilic mild/moderate Equine Asthma (N-mEA) or severe Equine Asthma (sEA), compared to healthy horses. We further hypothesize that increased airway cfDNA in horses with neutrophilic asthma will be associated with an increase in neutrophil extracellular traps (NETosis).

Over the past decade, equine research at North Carolina State University College of Veterinary Medicine (NCSU CVM) has grown exponentially under the leadership of Dr. Paul Lunn (Dean 2012-2022) and Dr. Kate Meurs (Associate Dean for Research and Graduate Studies date 2011- 2022; Dean 2022 - present). At present, our Equine Group is comprised of 16 faculty members with 1 additional search underway and 3 clinical veterinarians (See table below). Each member of our group has an active research program with group funding to date totaling over 45 million dollars. In addition, we are responsible for the training and specialty board certification of 5-6 equine residents/year, each of which must complete a research project of their own, as well as for the training of equine graduate (PhD and MS) students. While our group has been very successful at obtaining external funding (both federal and foundation) for our studies, we have struggled to maintain consistent funding for permanent NCSU CVM equine research herds, which are vital to all facets of our research and clinical work. Full funding for such herds is extremely difficult to fit within the limited budgets of most equine foundation grants. Additionally, funding for these herds is not directly applicable to the budget of many federal grants, whose proposed experiments rely on terminal studies or the use of clinical cases. However, the pilot data for these federal grants, which is absolutely essential for a successful application, does usually come from research herds. Lastly, horses in these herds, and particularly that of Dr. Schnabel???s research herd, are being used to make biologic products that have been highly successful for the treatment of equine hospital patients suffering from infectious arthritis and osteoarthritis as well as other ailments. While hospital revenue from such treatments helps to cover the costs of producing the biologic products themselves, it is not enough to cover the overall care and mandatory screening of these research herd horses for transmissible diseases. In addition to benefiting equine health and translational research, NCSU CVM equine research Herds also provide an important educational opportunity for NCSU students at all levels of training. Between them, Drs. Schnabel, Sheats and the Theriogenology group have trained over 40 undergraduate, 35 veterinary and 12 PhD students in research projects that rely on equine research herds. When appropriate, some of the research horses (Sheats) are also available for use in non-invasive equine science and veterinary teaching labs.

Equine asthma is diagnosed in horses of all breeds and sexes, with mild/moderate EA (mEA) reportedly affecting 60-100% of certain groups of horses, including young racehorses,1,2 and severe EA (sEA) reportedly affecting 10-20% of adult horses in the northern hemisphere.3???5 Characteristic symptoms of both mEA and sEA include recurrent and variable cough and exercise intolerance, with sEA horses showing signs of increased respiratory effort at rest.3???5 These symptoms occur when susceptible horses experience bronchoconstriction, mucus hypersecretion and airway inflammation as a result of exposure to organic dust.3???11 Routine treatments for EA include systemic and inhaled steroids and bronchodilators.12,13 Environmental management that reduces exposure to organic dust, such as increased pasture turn-out and elimination of hay, is the most effective long-term treatment for horses with EA.12,13 Without reduction of dust exposure, horses with sEA experience recurrent disease exacerbation. Unfortunately, elimination of organic dust from a horse???s environment is not always achievable for horse owners, leading to the need for long-term steroid treatment. However, long-term steroid treatment has drawbacks such as expense, if using inhaled medications, or laminitis risk and suppression of the hypothalamic-pituitary axis, if using systemic treatment.14,15 Therefore, alternatives to steroid therapy are needed for horses with EA.

Optimization of Equine Extracorporeal Therapy Protocol

Asthma is a syndrome of chronic airway inflammation that causes cough, shortness of breath, and chest tightness in 25 million Americans and is associated with 2.5 million deaths globally, each year. Compared to healthy individuals, people with asthma are at increased risk of adverse health effects caused by exposure to air pollution, specifically particulate matter ???2.5 ??m in diameter [PM2.5]. However, the cellular and biochemical mechanisms mediating immune dysfunction and exacerbation of underlying disease following exposure to air pollution remain largely unknown. This gap in knowledge is partly due to a lack of relevant translational animal models. Equine Asthma (EA) is a promising translational animal model for studies investigating the impact of PM-exposure on human respiratory health because EA is naturally occurring and has comparable pathophysiology to human disease, and equine and human monocytes/macrophages share similar biology. This proposal will use an in vitro PM-exposure model of alveolar macrophages acquired from healthy and asthmatic horses to identify differences in the immunophenotype, bioenergetic transcriptomic, and proteomic profiles of airway macrophages from healthy and asthmatic horses living full time outdoors, and determine the impact of PM-exposure on these responses. Findings from this study will further validate horses as an innovative and relevant model to study environmental impacts on human respiratory health. We will compare results of this study with results from our ongoing human studies funded by NIEHS to inform a new R01 application using the EA model to identify novel cellular targets to mitigate the negative impacts of PM-exposure on host immunity.

Investigating the Impact of Novel Amnion Extracts on Equine Intestinal Epithelial Cell Repair

Per- and polyfluoroalkyl substances (PFASs) are used to produce nonstick coatings, food wrappers, and fire-fighting foams. PFASs are environmentally persistent, ubiquitous and can be detected in the serum of 99% of Americans. Despite well-established immunotoxicity, few studies have investigated the effects of PFASs on the innate immune system. We propose combining primary human neutrophils and zebrafish larvae with innovative functional assays to investigate the impact of PFASs on neutrophil function and on the ability to recover from a bacterial infection. Our preliminary data reveal that two PFASs, PFHxA and GenX, suppress the ability of a neutrophil-like cell line to generate reactive oxygen species (ROS). Generation of ROS, along with phagocytosis, are hallmark features of activated neutrophils and play important roles during infection. We have also observed that these PFAS suppress the ability of zebrafish larvae to generate ROS, highlighting the potential of these compounds to negatively impact an organism????????s ability to recover from infections. In this proposal, primary human neutrophils will be exposed to PFHxA or GenX and their ability to generate ROS and undergo phagocytosis will be quantified. In addition, zebrafish larvae will be exposed to PFHxA or GenX, infected with a bacterial pathogen, and the bacterial burden quantified. We anticipate that these PFASs will suppress ROS generation and phagocytosis in primary neutrophils, and increase the bacterial burden in infected zebrafish larvae. Successful completion of this project will lay the groundwork for future research on deciphering the cellular and molecular mechanisms by which these PFASs suppress immune function.

Non-typhoidal Salmonellae are the leading cause of bacterial foodborne gastroenteritis and bovine food products are frequently implicated in disease outbreaks. The daunting rate of Salmonella infections in humans combined with the increasing number of multiple drug resistant (MDR) isolates is an alarming public health threat. MDR Salmonella infections are frequently traced to bovine sources, making it imperative to design novel, non-antimicrobial strategies to eliminate MDR Salmonella from cattle. In this basic science proposal, we will establish the role of a host-cell protein, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate), in Salmonella infections. We hypothesize that MARCKS protein function is essential for Salmonella-induced neutrophil recruitment and activation, epithelial invasion, and the overall survival of Salmonella in the host intestine. We will use a MARCKS peptide inhibitor to investigate the role of MARCKS in neutrophil antimicrobial responses in vitro and during calf infection. We will establish the role of MARCKS in Salmonella intestinal invasion in vitro and tissue colonization during calf infection. Finally, we will establish the role of our MARCKS peptide inhibitor on antimicrobial resistance development during calf infection. These experiments will (1) establish the basic biology of MARCKS protein during infection and (2) establish the efficacy of a MARCKS peptide inhibitor as a host-directed therapeutic alternative to treat bovine salmonellosis. Salmonella infections are a threat to both human and bovine health so alternatives to traditional antimicrobials are critically needed to combat this important pathogen. The proposed studies will directly inform development of a non-antimicrobial therapeutic to prevent and treat Salmonella in cattle.

Hypothesis: We hypothesize that upon hospital admission, plasma and/or peritoneal fluid cell-free DNA (cfDNA) will be significantly higher in horses with strangulating or inflammatory colic lesions vs. non-strangulating lesions, horses with SIRS score ???????????????2 vs. ???????????????1 (Roy et al. 2017), and/or nonsurvivors vs. survivors. Further, we hypothesize that admission levels of plasma and/or peritoneal fluid cfDNA will be positively correlated with admission levels of serum amyloid A (SAA) and lactate from the same sample type and/or total cost of care. Objective 1: To determine whether plasma cfDNA at hospital admission 1) is significantly higher in horses with strangulating or inflammatory vs. non-strangulating colic lesions, SIRS score ???????????????2 vs. ???????????????1, and/or survivors vs. nonsurvivors and 2) is correlated with admission SAA and blood lactate concentrations and hospital invoice. Objective 2a: To determine whether direct and/or diluted peritoneal fluid cfDNA values obtained with the Qubit 4 show a linear correlation to extracted peritoneal fluid cfDNA. Objective 2b: To determine whether peritoneal cfDNA at hospital admission 1) is significantly higher in horses with strangulating or inflammatory vs. non-strangulating colic lesions, SIRS score ???????????????2 vs. ???????????????1, and/or survivors vs. nonsurvivors and 2) is correlated with admission peritoneal SAA and peritoneal lactate concentrations and hospital invoice. Results from Objective 2a will be used to determine which type of peritoneal fluid sample to use for cfDNA measurement.

This project specifically investigates a novel non-antimicrobial target to combat enteric salmonellosis in cattle. Neutrophils play a key role in Salmonella enteritis, causing inflammation that ultimately enables Salmonella to colonize the gut. In this project, primary bovine neutrophils are used to assess how MARCKS protein (Myristoylated Alanine-Rich C-Kinase Substrate, a host protein known to regulate neutrophil functions) inhibition alters Salmonella-induced neutrophil inflammatory functions, such as adhesion, migration, and respiratory burst. Dr. Katie Sheat??????????????????s laboratory is currently conducting research to determine whether this MARCKS protein is a viable therapeutic target for combating neutrophilmediated tissue damage during diseases such as salmonellosis. The goal is that this translational research will benefit both livestock and humans by reducing the morbidity and mortality associated with enteric salmonellosis. Other potential applications of targeting the MARKS protein include the treatment of asthma and COPD across species.