Source: TEXAS TECH UNIVERSITY submitted to NRP
UNCOVERING MACROPHAGE AND ENDOTHELIAL CELL FUNCTIONAL DYNAMICS AND CROSSTALK IN PERIPARTURIENT DAIRY CATTLE ADIPOSE TISSUE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1031998
Grant No.
2024-67034-42234
Cumulative Award Amt.
$211,163.00
Proposal No.
2023-09720
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2026
Grant Year
2024
Program Code
[A1211]- Animal Health and Production and Animal Products: Animal Reproduction
Recipient Organization
TEXAS TECH UNIVERSITY
(N/A)
LUBBOCK,TX 79409
Performing Department
(N/A)
Non Technical Summary
For dairy cows, proper function of fat tissue is critical for supporting the health and productivity of these animals, especially at the onset of lactation when cows must deal with significant metabolic stress that can lead to inflammation, disease, and economic losses. Macrophages, the most abundant immune cells in fat tissue, and endothelial cells, which support blood flow into and out of fat tissue, are important cells that help guide the functions of fat tissue and play important roles in metabolism and inflammation. However, the changes that occur in these cell types and how they interact with each other as dairy cattle go from being pregnant to lactating, and when dairy cattle experience metabolic stress, have yet to be fully understood. Our hypothesis is that changes in both the abundance and functions of macrophages and endothelial cells within fat tissue of dairy cattle are involved in the development of metabolic stress and the adaptation required of dairy cattle during the transition from pregnancy to lactation.To investigate the macrophage and endothelial cells in fat tissue, we will collect fat tissue samples from dairy cattle both before and after calving as well as from dairy cows at the beginning of lactation that are experiencing metabolic stress. Macrophages and endothelial cells will be isolated from these fat tissue samples and evaluated for their overall abundance as well as potential changes in their function and genetics as cows transition from pregnancy to lactation. In addition, techniques will be used to understand how the macrophages and endothelial cells in fat tissue interact and how these interactions may differ during pregnancy and early lactation, as well as in cows that are experiencing metabolic stress. By furthering our understanding of the role that macrophages and endothelial cells play in the fat tissue of dairy cows, we may identify new targets for potential therapeutics for improving the health and productivity of dairy cattle during early lactation.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30534101030100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1030 - Cellular biology;
Goals / Objectives
For the training and development component of this project, the major goal is to prepare Dr. Hunter Ford for a career as an independent researcher. This goal contains the following objectives:Train Dr. Hunter Ford in the necessary research abilities and techniques for developing and engaging in future independent research projects.Develop Dr. Hunter Ford's professional skills and professional network so that he is a highly competitive candidate for potential job opportunities as an independent researcher.For the research component of this project, the major goal is to improve the health and productivity of dairy cows during the peripartum period by elucidating the roles of macrophages and endothelial cells in adipose tissue. In pursuit of this goal, this project contains two primary objectives:Characterize the transcriptomic and functional changes in macrophages and endothelial cells as dairy cows transition from late gestation to early lactation.Delineate the crosstalk between adipose tissue resident macrophages and endothelial cells in dairy cows with and without hyperketonemia in the early postpartum period
Project Methods
The training and development component of this project will focus on preparing Dr. Hunter Ford for a successful career as an independent researcher. Dr. Ford will receive primary mentorship and training from Dr. Clarissa Strieder-Barboza with support from the collaborating mentor, Dr. Carey Lumeng. Working in the Strieder-Barboza lab, Dr. Ford will receive extensive training in a wide range of techniques including adipose tissue biopsy and sample collection from dairy cows, transcriptomic analysis, flow cytometry, cell culture, cellular functional assays, and data analysis. Dr. Ford will also participate in the mentorship of undergraduate and graduate students and will continue the development of his teaching and communication skills by lecturing in courses associated with animal healthy, livestock immunology, and livestock disease. Dr. Lumeng will provide important technical expertise relating to adipose tissue macrophages and immunometabolism. Working with Dr. Lumeng, Dr. Ford will also present and discuss his research findings with members of the Lumeng lab. Dr. Ford will also participate in important professional development activities, including NIH workshops on flow cytometry, grant writing workshops, and career counseling sessions through Texas Tech University.Evaluation: Dr. Ford's training and development will be evaluated regularly via weekly one-to-one meetings with Dr. Strieder-Barboza, and quarterly with Dr. Lumeng. Dr. Ford's technical training and expertise will be assessed throughout this project on the basis of successful completion of experiments, responsible research practices, proper experiment design and data interpretation. Important milestones for Dr. Ford's training and development include successful completion of technical and grantsmanship workshops and career counseling sessions. Dr. Ford's development as a mentor will be evaluated based on the progress of the undergraduate and graduate students he is working with and will be measured through the successful publication of manuscripts co-authored by Dr. Ford and the students, as well as through successful presentation of research findings by student mentees at local and national conferences. Dr. Ford's teaching proficiency will be measured via student evaluations, as well as by other members of the Veterinary Sciences department at Texas Tech University.This research project will evaluate the adipose tissue resident macrophage and endothelial cells in dairy cattle using two cohorts of cows.The first cohort of cows will include 12 clinically healthy dairy cattle and subcutaneous adipose tissue samples will be collected from the flank of cows during both the late prepartum (-30 and -7 days relative to parturition) and early postpartum (+7 days relative to parturition) periods. Macrophages and endothelial cells will be isolated from these adipose tissue samples and will be evaluated for their abundance within adipose tissue as well as their transcriptomic and functional characteristics. Results will be analyzed via comparison between the three sampling dates. Using this cohort of dairy cattle, we will provide valuable insight into how changes within adipose tissue resident macrophage and endothelial cell populations contribute to the adaptation required of dairy cattle, and specifically the adipose tissue of dairy cattle, as these animals transition from pregnancy to lactation.The second cohort of cows will consist of cows between +4 and +10 days postpartum, 10 of which will be clinically healthy and 10 will have been diagnosed with hyperketonemia (blood BHB ≥ 1.2 mM). Hyperketonemia, a common sign that dairy cattle are dealing with significant metabolic stress, is largely driven by adipose tissue dysfunction, and is one of the most common metabolic diseases of early postpartum dairy cows. Subcutaneous adipose tissue will be collected from these cows after they have been selected for the study in the same manner utilized for the first cohort of cows. Adipose tissue resident macrophages and endothelial cells will be isolated and evaluated following the same procedures. In addition, macrophages and endothelial cells will be cultured and the resulting media from these cell cultures will be used to treat the opposing cell type (ex. Macrophage-conditioned media will be used to treat endothelial cells), with crosses of samples from healthy and hyperketonemic cows included as well (ex. Macrophage-conditioned media from macrophages isolated from hyperketonemic cows will be used to treat endothelial cells isolated from healthy cows). These indirect co-culture models will be used to evaluate the effects that the different cell types, within the context of metabolic disease, have on the function of each other. Overall, using this second cohort of dairy cattle will provide valuable insight into the roles of both macrophages and endothelial cells within adipose tissue in the development of metabolic disease during the early postpartum period.Efforts: Knowledge and findings pertaining to these studies will be disseminated through peer-reviewed publications and conference proceedings. Valuable insights will also be shared with local dairy farmers and incorporated into relevant classes for students studying immunology, dairy production, and metabolism.Evaluation: This project will be evaluated on a regular basis to ensure timely completion of the objectives. Important measurable milestones include the generation of transcriptomic data related to macrophage and endothelial cells populations and their publication in the NCBI Gene Expression Omnibus repository, presentation of findings in at least 2 academic research conferences per year, publication of at least 2 peer-reviewed research manuscripts, lectures in undergraduate and graduate level courses to discuss experimental findings, and presentations to colleagues and stakeholders via university meetings or on-farm discussions.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:In the first year of this fellowship, Dr. Ford's efforts reached the undergraduate and graduate students in Dr. Strieder-Barboza's lab via teaching and technical instruction on the methodologies and biology underlying the research goals of this fellowship. This included training on the processes of adipose tissue collection and processing, flow cytometry, cell culture, and experiment design. Dr. Ford presented results from the research conducted in the first year of this fellowship at the American Dairy Science Associated Annual Meeting in June 2025 to professional colleagues in the field of dairy science. Dr. Ford also worked with local dairy farmers and farm workers during the adipose tissue sample collections, explaining the purpose and methodology behind the research aims of the fellowship. Changes/Problems:The only major problemencounteredas part of the research supported by this fellowship pertains to the adipose tissue macrophagesisolation process.A lower number of macrophages were successfully isolated from the adipose tissue of dairy cows than what was expected; however, no major changes in the research aims of this fellowship wererequired.To ensure the reliability and validity of the protocols and methodologies for evaluating macrophage transcriptomic profiles and macrophage-endothelial cell crosstalk,Dr. Ford performed many of theinitialstudies using peripheral blood mononuclear cells (PBMCs) isolated from the same cows as the adipose tissue samples were obtained.While not specific to adipose tissue, theseinitialstudiesprovidedvaluable insight into potential immune cell-endothelial cell crosstalk in dairy cows with and without hyperketonemia andprovidedDr. Ford the opportunity tooptimizethe experiments beforeutilizingthe limited macrophages. Theinitialresults from the PBMC-endothelial cell experiments were presented by Dr. Ford at the American Dairy Science Association Annual Meeting in June 2025. Dr. Ford plans to conduct experiments withtheadiposetissuemacrophages in the coming months. What opportunities for training and professional development has the project provided?Dr. Ford hasparticipatedin professional development events hosted by the Obesity Research Institute at Texas Tech University as well as at the American Dairy Science Association Annual Meeting in June of 2025. Both eventsprovidedDr. Ford the opportunity to interact and network with colleagues in both industry and academia, gaining insight into what type ofexpertise, knowledge, and soft skills will support his competitive candidacy for job opportunities in the future. Dr. Ford hasworked closely with both undergraduate and graduate students in Dr. Strieder-Barboza's lab, providing mentorship on laboratory technical skills and knowledge of the biology supporting the aims of this fellowship. Dr. Ford has also prepared and taught lectures on macrophage biology and immunology in the graduate student course"Application of Immunology in Animal Health", as well as classes on periparturient dairy cow diseasesin the graduate student course "Animal Diseases in Livestock". How have the results been disseminated to communities of interest?In the first year of this fellowship, Dr. Ford has disseminated the results of his research efforts to undergradute and graduate students at regular lab meetings with the Strieder-Barboza lab. Key results were also presented to academic colleagues and industry professionalsat the American Dairy Science Associated Annual Meeting in June 2025. What do you plan to do during the next reporting period to accomplish the goals?The first aimof the researchcomponentof this fellowship is scheduled to start in January 2026. This will involve repeated adipose tissue biopsies in dairy cows as they transition from late gestation to early lactation. Many of the same experiments and analyses that Dr. Ford has been conducting with the samples obtained during the project for the second aim will be repeated for these samples, with a focus on the role of adipose tissue macrophages and endothelial cells in the metabolic changes that occur in dairy cows over this critical time period. Dr. Ford expects biopsies to be concluded by mid-March. Dr. Ford's professional development will continue duirng the next reporting period. Dr. Ford will prepare and teach classes in the graduate student courses"Application of Immunology in Animal Health" and "Animal Diseases in Livestock". Dr. Ford will attend professional development seminars offered by Texas Tech University focused on preparing competitive applications for roles in both industry and academia.

Impacts
What was accomplished under these goals? Dr. Hunter Fordhas continuedhis technical and professional development during the first year of this postdoctoral fellowship. Dr. Ford has become proficient in multiple techniquesdirectly relatedto the research aims of this project, as well as techniques that can beutilizedin a wide range of research investigations that may be of interest in the future. Dr. Ford has developed technical skills and methodologies for understanding endothelial celland macrophagefunction in adipose tissue of dairy cows, including flow cytometry and endothelial celland macrophageisolation,tubulogenesis, and permeability. Dr. Ford also developed methods for understanding endothelial cell - immune cell crosstalk, using a conditioned media approach to study the effects of immune cellsecretomeson endothelial cell function. Dr. Ford has also worked to improve and refine his bioinformatics capabilities andexpertise. Dr. Ford analyzed the transcriptomic profiles of the endothelial cells isolated during the first part of this fellowship and plans to repeat this process with the macrophages. Dr. Ford has also expanded his bioinformatics abilitiesin the area ofsingle-nuclei RNA sequencing analysis; working with both dairy cow and human datasets in collaboration with his mentor, Dr. Clarissa Strieder-Barboza. Dr. Ford successfully published themanuscriptpertaining tothe dairy cow data in September 2025, with plans to publish at least two other first-author single-nuclei/single-cell RNA sequencing manuscripts over the next year. In the first year of this project, experiments werefocused onaim2. The main goal was to definethe role and crosstalk of macrophages and endothelial cells in the adipose tissue of dairy cows with and without hyperketonemia during the early postpartum period. Adipose tissue biopsies and cell isolations occurred in the Fall of 2024.Dr. Ford has been running the associated studies. For better understanding the endothelial cell populations, Dr. Fordhas analyzed the transcriptomic profiles of the adipose tissue endothelial cells from the two groups of cows used in this study. Initial analysis has revealed a potentially suppressed capacity for vascular remodeling and an increased immune/inflammatory profile in the adipose tissue endothelial cells fromhyperketonemicdairy cattle. Working with adipose tissue macrophages has proved to be a challenge, primarily due to thelow numberof macrophagesisolatedvia flow cytometry cell sorting (addressed in changes/problems). Dr. Ford has been working tooptimizeprotocols and methods for transcriptomic analysis of macrophages and macrophage-endothelial crosstalk investigations to ensure that experiments can be properly conducted with the limitednumberof macrophages obtained so far. Dr. Ford plans to conduct these experiments and obtain the final data from this part of the fellowship research plan in the coming months.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Ford, H., Strieder-Barboza, C. Depot-dependent effects of subclinical ketosis on visceral and subcutaneous adipose tissue transcriptional cellular diversity in dairy cows. J Animal Sci Biotechnol 16, 128 (2025). https://doi.org/10.1186/s40104-025-01265-y
  • Type: Conference Papers and Presentations Status: Published Year Published: 2025 Citation: Ford, H. S., Bridger; Machado, Vinicius; Nelson, Olivia; Beneduzi, Natalia; Benitez Rojas, Oscar; Strieder-Barboza, Clarissa (2025). Effects of PBMC-conditioned media on visceral adipose tissue endothelial cells transcriptome and proliferation in early postpartum dairy cows with ketosis. American Dairy Science Annual Meeting 2025, Louisville, Kentucky, United States, Journal of Dairy Science.