Abstract
Neutrophilic inflammation is a critical characteristic in many pulmonary diseases. However, current preclinical screening methods in high-throughput platforms typically fail to capture critical neutrophilic pathophysiology including blood-to-lung recruitment, dysfunctional activation, and the impacts on the air-blood barrier. These limitations thus hinder therapeutic development and investigation for these diseases. In order to balance the demands between physiological complexity and high-throughput, we developed an air-blood barrier model incorporating with human neutrophils in a 96-well platform to demonstrate neutrophil recruitment and functional phenotypes under inflammatory conditions. We modeled inflammatory distal airway with epithelial-side interleukin 8 (IL-8) and found dose dependent reduction of recruitment with baricitinib, a JAK1/2 inhibitor, within physiologic doses. Additionally, neutrophil recruitment to cystic fibrosis patient-derived sputum supernatant induced the recruitment, disease-mimetic phenotypic shift of healthy donor neutrophils, and upregulated endothelial e-selectin. With this high-throughput model, required sample volumes are reduced by 25 times per well compared to 24-well assays with quadruplicate per plate. Despite the small sample volume utilized in this model, sufficient number of recruited neutrophils are obtained for downstream flow cytometry. This novel high-throughput in vitro lung air-blood barrier model combined with leukocyte recruitment and phenotyping advances opportunities for pathophysiological studies, personalized medicine, and drug testing applications.

"Single-cell RNA Sequencing to Identify Early Systemic and Local Immune Biomarkers of Dysregulation Following Severe Musculoskeletal Trauma" - Drishti Maniar, Ph.D. Student, Krish Roy Lab

Abstract
Severe musculoskeletal trauma is a major clinical concern, accounting for up to 78% of non-fatal trauma-related injuries. These injuries often lead to serious complications, with bone nonunion in long-bone fractures being particularly challenging. Post-traumatic bone nonunion results in significant clinical challenges, including extended hospital stays, multiple surgeries, potential limb amputations, and in severe cases, mortality. Following severe trauma, both local tissue damage and systemic responses contribute to immune dysregulation. This dual impact manifests locally at the injury site and systemically, characterized by alterations in immune cell populations, including increased myeloid-derived suppressor cells (MDSCs). Despite recognition of these immune changes in trauma survivors, the early cellular and molecular mechanisms underlying both local and systemic immune dysfunction remain poorly understood. This knowledge gap has impeded the development of effective interventional strategies to restore immune homeostasis and improve functional bone regeneration. We propose using single-cell RNA sequencing to analyze both systemic blood and local tissue immune microenvironments in a pre-clinical rat trauma model during the first week post-injury. Using approaches including differential expression gene analysis, gene set enrichment analysis, and cell-cell interactions, we aim to identify early biomarkers of immune dysregulation and mechanisms driving pathogenesis at both systemic and local levels. This research will reveal therapeutic targets for early intervention strategies, and we utilize these insights to develop immunomodulatory nanoparticles to target cell populations involved in trauma pathogenesis.