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Item Abstract 16: Insights into Highly Engraftable Hematopoietic Cells from 27-Year Cryopreserved Umbilical Cord Blood(Oxford University Press, 2023-09-04) Broxmeyer, Hal; Luchsinger, Larry; Weinberg, Rona; Jimenez, Alexandra; Masson Frenet, Emeline; van't Hof, Wouter; Capitano, Maegan; Hillyer, Christopher; Kaplan, Mark; Cooper, Scott; Ropa, James; Microbiology and Immunology, School of MedicineIntroduction: Cord blood banking has consistently outpaced the utilization of cord blood units (CBUs). Thus, the average duration of cryopreservation among banked CBUs will likely continue to increase. It remains unclear how long cryopreserved CBUs remain functional, and it is critical to determine whether duration of cryopreservation should be used as an exclusionary criterion during selection for clinical use or if alternative post-thaw metrics can identify potent cryopreserved CBUs regardless of age. Objectives: Our goal was to determine whether long-term (27-year) cryopreserved CBUs retain viable and functional hematopoietic stem (HSCs) and progenitor cells (HPCs). We further sought to leverage differences in HSC/HPC function (measured by in vivo engraftment) to demonstrate the utility of using omics approaches to identify candidate genes for use as molecular potency markers. Methods: We performed comprehensive ex vivo, in vivo, and molecular analyses on the numbers, viability, and function of three 27-year cryopreserved CBUs using 3-year cryopreserved and fresh CBUs for comparison. Assays included viability staining, immunophenotyping by flow cytometry, primary and secondary colony forming unit (CFU) assays, ex vivo expansion of immunophenotypic HSCs/HPCs/CFUs, limiting dilution transplantations into immune-deficient mice, secondary transplantations, and RNA-sequencing of sorted HSCs and multipotent progenitor cells. Results: Compared to fresh and recently cryopreserved CBU controls, long-term cryopreserved CBUs yield statistically similar numbers of viable immunophenotypic HSCs, multipotent HPCs, and committed myeloid and lymphoid HPCs. They retain highly functional cells, demonstrating similar primary and secondary CFU numbers and expansion capacity compared to controls, as well as robust engraftment, SCID repopulating cell frequency, and secondary engraftment capacity in mouse models of transplantation. Transcriptomic modelling revealed 18 genes, including MALT1 and MAP2K1, and several gene programs, including lineage determination programs and oxidative stress responses, that are strongly enriched in high engrafting HSCs/HPCs. Discussion: CBUs cryopreserved for up to 27 years retain highly functional HSCs/HPCs. Thus, duration of cryopreservation alone is not an ideal exclusionary criteria for selection of CBUs. Preserving older CBUs may help to maintain a large and diverse pool of donors for clinical selection. Further, transcriptomics can identify candidate genes associated with engraftment for elucidation of possible CBU potency markers regardless of the duration of cryopreservation.Item Abstract 26: The Role of Oxygen in Cord Blood Hematopoietic Stem and Progenitor Cell Expansion and Engraftment(Oxford University Press, 2023-09-04) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Kaplan, Mark; Capitan, Maegan; Microbiology and Immunology, School of MedicineIntroduction: Hematopoietic stem (HSC) and progenitor cells (HPCs) are exposed to differing oxygen tensions ranging from <1% to 21% as they reside in/move through different tissues or are harvested for clinical utility. Functional changes in HSCs/HPCs are induced by acute changes in oxygen tension (e.g., a change in percent of cells in cycle). Objectives: We sought to determine if variable oxygen levels affect expansion and/or functional properties of cord blood (CB) HSCs/HPCs ex vivo and in vivo. Methods: Human CB CD34+ cells were grown in expansion culture +/-UM171, an agonist of HSC self-renewal that expands transplantable CB HSCs, in five oxygen tensions: 1%O2, 3%O2, 5%O2, 14%O2, and 21%O2. HSCs/HPCs were enumerated by flow cytometry. Functional HPCs were enumerated by plating in semi solid media for colony forming unit assays (CFU). Cell cycle and reactive oxygen species (ROS) were measured by flow cytometry. Ability of expanded cells to engraft was determined by transplantation in non-lethally irradiated NSG mice. Results: Immunophenotypic HPCs and functional HPC CFUs expanded significantly more after 7 days of growth in higher oxygen tensions (5%O2-21%O2) compared to lower (1%O2-3%O2), while immunophenotypic HSCs expanded best at 5% O2. HSCs/HPCs grown in low oxygen tensions had significantly lower ROS levels, significantly higher percentage of cells in G0, and were slightly but reproducibly smaller/less granular than those grown in high oxygen levels. HSC/HPC numbers were reduced in high oxygen tensions 1-2 days after plating but were better maintained in low, suggesting cells undergo a culture shock/stress after plating that is mitigated by reduced oxygen. In the presence of UM171, HSCs expanded significantly better at higher oxygen levels, but HPCs are better maintained in 5%O2. Ex vivo CD34+ expansions maintained under physiological O2 levels (1-14%O2) demonstrated significantly better/faster neutrophil recovery following transplantation compared to cells expanded at 21%O2 or input. Discussion: HSCs/HPCs proliferate rapidly in high oxygen but have fewer quiescent cells, higher ROS, and are larger and more granular which are all characteristics associated with exhaustion. While high oxygen allows for faster growth, low tensions may mitigate cell stress and allow for prolonged growth (i.e., HSC/HPC expansion) while maintaining functional properties.Item Abstract 33: Transcriptomic Identification of Functionally Potent Umbilical Cord Blood Units(Oxford University Press, 2024-08-21) Ropa, James; Gutch, Sarah; Beasley, Lindsay; Van't Hof, Wouter; Sun, Jessica; Capitano, Maegan; Kaplan, Mark; Medical and Molecular Genetics, School of MedicineIntroduction: Umbilical cord blood (UCB) is an important donor source for standard of care cellular therapies as well as innovative new treatments. Universal potency criteria for cord blood unit (CBU) selection for different cellular therapy applications are still desired and efficient methods to elucidate these criteria remain elusive. Objectives: Our goal is to find molecular markers that identify potent CBUs for use in cellular therapies. Here, we utilized transcriptomics to reveal genes associated with hematopoietic stem and progenitor cell (HSC/HPC) potency in hematopoietic cell transplantation. Methods: We performed three separate transcriptomic analyses of human UCB used in mouse models of transplantation. This included bulk RNA-sequencing of HSCs/HPCs from CBUs with known engraftment capacities (n=9 CBUs), bulk RNA-sequencing of homed/early engrafted CD34+ cells (n=3 CBUs), and single cell RNA-sequencing of CD34+ cells expanded in varying oxygen tensions, which affects their transplantation potency (n=4 CBUs). Results: HSCs/HPCs enriched for dehydrogenase and cell cycle associated genes yield better repopulating cell frequency. Early homed CD34+ cells have enriched expression of immune activation and cell cycle genes compared to input transplanted cells. Distinct clusters of UCB cells marked by genes such as PRSS2 and AVP are enriched in oxygenation conditions that drive increased potency. Dehydrogenase and stress response genes are enriched in populations predicted to be more functional regardless of HSC/HPC subpopulation. Integration of all three studies reveals genes that may define highly potent CBUs, including DDIT4, a stress response gene. Indeed, DDIT4 independently predicts engraftment outcomes in mouse models of transplantation. Future work will examine a qPCR based gene panel potency assay to predict outcomes in patient transplantations. Discussion: We have identified genes associated with HSC/HPC potency using transcriptomic approaches. These findings have immediate translational implications for CBU selection for transplantation, but also provides a blueprint for finding CBUs best suited for use in developing off-the-shelf immune effector therapies or those that are best for treating non-hematologic central nervous system disorders such as cerebral palsy, among other applications. Importantly, this study highlights the importance for omics technology as a valuable tool to define potency criteria for UCB as a donor source for cellular therapies.Item Alternative Splicing Profile Comparison of Differentiating I-helper Cells to Dissect the Splicing Signatures of Th1, Th2, 1h17 and Treg CellsLakshmipati, Deepak Kumar; Quoseena, Mir; Ulrich, Benjamin; Kaplan, Mark; Janga, Sarath ChandraThis study focuses on the contribution of Alternative Splicing (AS) events in the differentiation and post-differentiation functions of T-helper cells, specifically in Thl, Th2, Th9, Th17 and Treg cells. T cell RNA-seq data from 72hr and 2week post differentiation time points was analyzed using (r-MATS) for alternative splicing events. We observed majority of the significant events are Skipped Exon (SE) events originating from a total of 1,556 genes and lntron Retention (RI) events were the second most abundant event occurring in 1,254 genes at 72 hours post differentiation. These numbers were significantly lower at 2 weeks post differentiation. PCR and qPCR validations confirmed scores of novel splicing event predictions. Results showed several skipped exon (SE) events in KTNl, IL4RA IL27, Hnrmpd, CREM and Arid4b showing different mRNA isoforms across multiple naïve vs differentiated T cell combinations. Overall, RI event associated genes were more prevalent (3,239 genes) than those exhibiting SE (2810 genes). SE events were associated with 10.8% (Th17), 11.2% (Treg), 12.1% (Th2) and 13.9% (Thl) of the genes, a similar trend was observed with RI events with a prevalence of 12.2% (Th17), 12.5% (Treg), 14.2% (Thl) and 14.4% (Th2) of the genes. Gene ontology results showed most of the genes showing SE and RI events are involved in processes like 'mRNA Processing', 'RNA Processing' and 'RNA Binding' and ontology results for retained introns also showed p53 suppression proteins, regulated exocytosis of neurotransmitters and hormones. It was also observed that Introns consistently favored retention at the 3' end of the gene than the 5', with 430 genes showing intron retention events at the 3' end and 21 genes exhibiting them at the 5' end, for the 72 hour time point. Enriched functional ontologies were consistently seen across all cell types to be exclusive for the genes showing RI in the 5' end vs the 3' end.Item Award Recognition for "Developing a Minimally Invasive Cell-Based Model to Predict Response to Major Trauma"(2021-08) Gates, Kayla L; Nunge, Rebecca A; Adom, Jamila; Kaplan, Mark; McKinley, ToddBackground: The physiologic response to injury is heavily influenced by the immune system. The complexities of the immunologic response to injury are becoming increasingly understood as researchers have leveraged computational methods that allow temporal and spatial coordination of immune mediator orchestration to be quantified. Recently, early differences in immunologic orchestration have been shown to stratify individual tolerance to injury. Specifically, there are subsets of trauma patients that are either sensitive or tolerant to hemorrhage that demonstrated notably different early immunologic orchestration of mediators from clusters of cytokines that are primarily tissue protective or pro/anti-inflammatory. These differentiating networks of mediators formed and dissipated over the initial 72 hours after injury clearly demonstrating that the immunologic response to injury is an acute dynamic event that has pathomechanistic relevance to outcomes after injury. Additionally, it is distinctly possible that individualized differences in immune response may determine tolerance/sensitivity to injury. The differential immunologic response to trauma represents an opportunity to discover specific factors that may be predictive of a patients’ response to traumatic injury and subsequent hemorrhagic shock. Accordingly, we have embarked on a line of experimentation to explore potential precision approaches based on individual immunologic response to injury. Here we report our initial experimental findings in conceptual model development with the ultimate goal of developing minimally invasive/non-injurious testing that will accurately identify individual tolerance to hemorrhage and injury. In this experiment, an in-vitro cell-based assay was designed to mimic traumatic injury. Specifically, we tested the immunologic response in murine splenocytes to a simulated hypoxic injury, a simulated mechanical injury and a simulated open wounding injury. The development of a reliable cell-based model will allow investigation to determine correspondence and relevance between cell-based responses to non-traumatic injury and in vivo immunologic response to trauma, with the overall goal of developing a reliable test to predict response to traumatic injury in humans. Methods: In-vitro cellular responses of murine splenocytes are reflective of peripheral blood cell responses and were used for pilot experiments. Splenocytes from C57BL/6 (B6) BALB/c or CH3/ HeJ strains mice were used with stimuli that mimic traumatic injury using chemical (hypoxia or sepsis) or mechanical (shear stress) stimuli that might from an open wounding type of injury. Hypoxia was simulated by subjecting cell cultures to hydrogen peroxide. Sepsis was simulated by subjecting cells to lipopolysaccharide (LPS). Some culture conditions included several individual cytokines associated with acute inflammation and external pathogens (interleukin (IL)-6, IL-1β, IL- 33), the damage molecule high mobility group box protein (HMGB)-1, or combinations of LPS and the cytokines. Following treatment, cDNA was prepared and used for qPCR amplification of TNFα, HIF1α, and BAX to assess inflammation, hypoxia, and apoptosis, respectively. Multiplex analysis of IL-21, IL-4, IL-22, IL-5, and IL-10 expression was performed from culture supernatant collected at 24 hours after stimulation. Flow cytometry was performed to assess proliferation of immune cells following treatment. ELISA was conducted to quantify production of the cytokine IL-9 that occurred following splenocyte stimulation. Results: Analysis of C57BL/6 splenocyte viability show that any combination of cytokines or LPS did not impact cell survival, while hydrogen peroxide reduced survival significantly in each treatment group. From the qPCR data, LPS generated a 4x increase in TNFα expression relative to control, while cytokine treatment yielded no expression changes. Treatment with LPS + cytokines closely resembled the LPS treatment group. LPS treatment reduced expression of HIF1α, while hydrogen peroxide increased expression of HIF1α. The addition of cytokines reduced expression of HIF1α in groups that were treated with both hydrogen peroxide and cytokines. ELISA analysis of the proinflammatory cytokine IL-9 indicated increased production of IL-9 following treatment with LPS + cytokines. In the second experiment, the model was applied to three different strains of mice in order to gauge differences in the immune response to the same cellular stress. Multiplex analysis showed no significant changes in IL-4 or IL-21 expression in any of the strains. C3H mice showed no response to LPS, which was expected due to LPS resistance in these strains. In the B6 and BALB mice, IL-10 was induced by LPS treatment. BALB mice also showed increased expression of IL-5 and IL-22 in response to mechanical stress, while the other strains showed no response. IL-10 expression was not induced by mechanical injury in any strain. Flow cytometry analysis was used to assess immune cell response to stimuli. Both B6 and C3H mice showed increased percentages of CD4 and CD8 cells in response to mechanical stimulus, LPS, and LPS + cytokine treatment relative to control. Macrophage levels were more elevated in B6 mice in response to mechanical stimulus, whereas levels decreased in the C3H mice. Discussion: The overall goal of this line of investigation is to develop minimally invasive and non-injurious testing that can be used to determine individual tolerance/sensitivity to trauma and hemorrhage. These pilot studies were used to determine how immune cells can be isolated and stimulated to mimic injury. Splenocytes were used as they encompass a broad cross-section of white blood cells. Clear inter-strain differences were evident between the B6, BALB and C3H mice. Hypoxia stimuli consistently resulted in roughly a 50% loss of cell viability and accordingly may not be a viable strategy. The greatest effects were encountered with LPS +/- addition of stimulating cytokines. We measured changes in five of six cytokines in B6 mice and four of six BALB mice involving reparative cytokines (IL-21 and 22), anti-inflammatory cytokines (IL-10) and in type 2 cytokines (IL-4 and IL-5). Accordingly, these strains and stimulation methods will be expanded to determine effects on production on a broader panel of cytokines. In addition, computational methods will be leveraged on the next experiment to determine in-vitro effects on immunologic mediator orchestration to account for time-dependent mediator networks and spatial networks of mediators. Moving forward, these experiments will be repeated to reproduce our findings and improve our ability to distinguish between varying immune responses. Results will then be paired with studies examining the responses to traumatic injury among these and other strains. The overall goal of this project is to accurately predict the response to an in-vivo injury using an in-vitro non traumatic stimulus. Findings from this project will enable the development of a clinical test that accurately predicts immunologic response to trauma and stratify individual tolerance to hemorrhage and injury.Item Biological Functions of Intracellular Hepatitis B e Antigen(2019-09) Mitra, Bidisha; Guo, Haitao; Androphy, Elliot J.; Kaplan, Mark; Yu, Andy; Lu, TaoThe function(s) of the intracellular form of HBeAg, previously reported as the preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we propose to elucidate the translocation of p22 during its formation from endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have identified that a portion of p22, after the cleavage of its signal peptide in ER, is released back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except for the extended 10 amino acid precore region at the N-terminus, we observed that p22 wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not the secreted HBeAg significantly reduced interferon stimulated response element (ISRE) activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN- α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1, indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus evade host innate immune response and cause resistance to IFN therapy in patients. Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results indicate that there is a novel ER sorting mechanism for the distribution of the intracellular and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism.Item Characterization of Allergen-Specific Immunoglubulin E Development in a Food Allergy Model and Its Regulation by T Follicular Helper and T Follicular Regulatory Cells(2023-05) Chen, Qiang; Dent, Alexander; Kaplan, Mark; Brutkiewicz, Randy; Zhou, BaohuaFood allergy is a highly prevalent and serious disease regulated by immunoglobin E (IgE) antibodies specific for food allergens.The development of IgE is regulated by T follicular helper cells (TFH) and T follicular regulatorycells (TFR) in the germinal center (GC). We aimed to understandthe regulation of IgEin the GC by TFH and TFR cellsusinga mouse food allergy model. We found that the dosage and timingof allergen delivery into thegut is criticalfor allergen-specific IgE development, in part because the timing of allergen delivery affected the expression of regulatory factors by TFH and TFR cells. We studied FGL2, an inhibitory factor, and found that down-regulation of FGL2 in TFH cells was important for the allergic IgEresponse. Apart from inhibitory factors, TFH cell-derived IL-4 is required for IgE responses. We unexpectedlyfound that TFR cells in food allergy produce comparable amountsof IL-4 to TFH cellsand IL-4–expressing TFR cells promoteallergen-specific IgEin food allergy. The IgE response is highly sensitive to IL-4 levels, suggesting the need for extra IL-4 from TFR cells. However,TFR cells have distinct functionsdepending on the immune environment, since TFR cells repress IgEinanairway inflammation model. We found that TFR cells in airway inflammation have a different gene expression profile from TFR cells in food allergy, whichmay explain their distinct functions. Lastly, previous studies showed that high-affinity IgE driving anaphylactic reactions is produced via IgG1-switchedintermediate B cells. We challenged this paradigm by showing that high-affinity IgE develops in the absence ofIgG1-switchedB cellsin our food allergy model.Overall, our studies reveal that IgE is regulated by novel pathways in food allergy. We hope to exploit these new pathways to develop new specific therapies for food allergy.Item Developing a Minimally Invasive Cell-Based Model to Predict Response to Major Trauma(2020-07-31) Nunge, Rebecca A; Gates, Kayla L; Adom, Jamila; Kaplan, Mark; McKinley, ToddBackground. Physical trauma results in a systemic inflammatory response. Preliminary research in orthopedic trauma patients suggests that patients with similar demographics and severity of injury vary in their response to traumatic injury. Analysis of the immunological response post-injury showed a sustained pro-inflammatory response with delayed reparative cytokine expression in trauma sensitive patients, while the trauma tolerant patients had an early inflammatory expression with resolution by 72 hours post-injury. Thus, we hypothesize that differential response to non-traumatic injury might serve as a predictive tool for the identification of trauma tolerant and sensitive patients prior to injury. The goal of this research is to test whether immunological changes to inflammatory stimuli can predict tolerance or sensitivity to trauma using an-vitro cell-based assay. Methods. Splenocytes were isolated from naive C57BL/6 mice and subjected to biological trauma in vitro using LPS (100 ng/mL) or hypoxic trauma using hydrogen peroxide (50 µM, 100 µM, and 200 µM) with or without proinflammatory cytokines, IL-1β (1 ng/mL) , IL-6 (200 ng/mL), and IL-33 (150 ng/mL). Inflammation and hypoxia were assessed using IL-6 and HIF-1ɑ expression respectively via qPCR 24 hours post-treatment. Cell death and pro-inflammatory cytokine production using multiplex analysis were used to measure outcomes. Results. Both types of treatments showed increased cell death compared to the control group. qPCR data is pending. Conclusion. With these studies as a core of the experimental approach, this in vitro cell-based assay will be used to assess immunologic response to inflammatory stimuli across the genetic variation of mouse strains. Findings from this project could enable the development of a clinical test that accurately predicts immunologic response to trauma and related-complications based on patients’ sensitivity to pre-traumatic injury.Item The IUPUI Signature Center for Atopic Dermatitis(Office of the Vice Chancellor for Research, 2010-04-09) Travers, Jeffrey B.; Kaplan, Mark; Holbreich, Mark; Leickly, FrederickAtopic dermatitis is a chronic inflammatory skin disease characterized by dry skin, hypersensitivity to irritants and allergens, and significant pruritus. Atopic dermatitis is commonly associated with other atopic diseases including asthma, allergic rhinitis, and gastrointestinal disorders including eosinophilic esophagitis. Though a very common disease, there exists much misinformation and controversy/conflict in both the lay and medical communities about its pathogenesis and treatment, resulting in suboptimal care for the atopic dermatitis patient. Thus, education of both clinicians and lay public is needed. Inasmuch as atopic dermatitis is considered a systemic disorder, the optimal management should entail a multidisciplinary approach. Finally, research into the mechanisms by which atopic dermatitis occurs is needed to improve treatment of this common and quite debilitating disorder. The objective of the IUPUI Signature Center for Atopic Dermatitis is to provide optimal patient care, education and research in atopic dermatitis for the citizens of the state of Indiana. The Atopic Dermatitis has three separate components. First, we have developed an Atopic Dermatitis Working Group (ADWG) consisting of clinicians and scientists who meet on a monthly basis to disseminate information about research ideas/trials, and discuss topics and present difficult patients. Second, we have developed a monthly multidisciplinary AD clinic which has attracted the most challenging AD patients. Finally, we have developed infrastructure to assist in clinical and basic science research projects involving AD. Altogether, the IUPUI Signature Center for AD has been very successful as measured by the numbers of clinicians, researchers and patients who have been impacted by its presence.Item Understanding the Integrated Pathophysiological Role of a Moonlighting Protein in Lung Development(2019-08) Lee, Dong Il; Schwarz, Margaret; Tune, Johnathan; Kaplan, Mark; Basile, DavidSensing, integrating, and relaying signals from the environment through proteins, metabolites, and lipids to the lung are critical for proper development. Moonlighting proteins, such as AIMP1, are a unique subset that serves at least two independent physiological functions. Encoded by gene AIMP1, AIMP1 has two known functions: (1) C-terminus EMAP II domain of full-length AIMP1 can be secreted out of the cell to chemoattract myeloid cells; (2) intracellular full-length protein interacts with tRNA synthetases in protein translation. However, despite the linkage of protein expression levels of with several lung pathologies such as bronchopulmonary dysplasia (BPD), effectively targeting the protein encoded by AIMP1 has been a challenge due to poorly understood mechanisms. This thesis explores physiological, signaling, and immunological moonlighting mechanisms of first, the extracellular EMAP II then the intracellular AIMP1. Experiments utilize both in vitro and in vivo models, including a murine model of BPD and Cre-mediated exon-deletion knockout. Experimental results provide evidence that in the BPD model, EMAP II levels are elevated and sustained – first in bronchial epithelial cells then in macrophages. Mice exposed to sustained and elevated EMAP II protein levels resemble the BPD phenotype while neutralization partially rescued the phenotype, implying EMAP II as a potential therapeutic target against BPD. Results from studies exploring EMAP II’s signaling mechanism identify transient stimulation of JAK-STAT3 phosphorylation, commonly found in inflammation-resolving macrophages. In contrast, it induces unique transcriptional changes that are reversible both by JAK-STAT inhibitor and siRNA-mediated knockdown of Stat3. Studies using AIMP1 knockout mouse reveal a novel function for the intracellular AIMP1. AIMP1 knockout mice exhibited neonatal lethality with a respiratory distress phenotype, decreased type I alveolar cell expression, and disorganized bronchial epithelium, suggesting a role in lung maturation. In vitro experiments suggest that a portion of AIMP1 residing in the cell’s membrane interacts with various phosphatidylinositols and contributes toward F-actin deposition and assembly. Data from these experimental studies provide insight into how the various functions of the promiscuous AIMP1 gene affect lung development. These studies exemplify not only characterize novel moonlighting mechanisms of AIMP1, but also highlight the importance of characterizing moonlighting proteins to promote therapeutic preventions.