Modeling The Spatiotemporal Dynamics Of Cells In The Lung

Multiple research problems related to the lung involve a need to take into account the spatiotemporal dynamics of the underlying component cells. Two such problems involve better understanding the nature of the allergic inflammatory response to explore what might cause chronic inflammatory diseases such as asthma, and determining the rules underlying stem cells used to engraft decellularized lung scaffolds in the hopes of growing new lungs for transplantation. For both problems, we model the systems computationally using agent-based modeling, a tool that enables us to capture these spatiotemporal dynamics by modeling any biological system as a collection of agents (cells) interacting with each other and within their environment. This allows to test the most important pieces of biological systems together rather than in isolation, and thus rapidly derive biological insights from resulting complex behavior that could not have been predicted beforehand, which we can then use to guide wet lab experimentation. For the allergic response, we hypothesized that stimulation of the allergic response with antigen results in a response with formal similarity to a muscle twitch or an action potential, with an inflammatory phase followed by a resolution phase that returns the system to baseline. We prepared an agent-based model (ABM) of the allergic inflammatory response and determined that antigen stimulation indeed results in a twitch-like response. To determine what might cause chronic inflammatory diseases where the twitch presumably cannot resolve back to baseline, we then tested multiple potential defects to the model. We observed that while most of these potential changes lessen the magnitude of the response but do not affect its overall behavior, extending the lifespan of activated pro-inflammatory cells such as neutrophils and eosinophil results in a prolonged inflammatory response that does not resolve to baseline. Finally, we performed a series of experiments involving continual antigen stimulation in mice, determining that there is evidence in the cytokine, cellular and physiologic (mechanical) response consistent with our hypothesis of a finite twitch and an associated refractory period. For stem cells, we made a 3-D ABM of a decellularized scaffold section seeded with a generic stem cell type. We then programmed in different sets of rules that could conceivably underlie the cell\u27s behavior, and observed the change in engraftment patterns in the scaffold over selected timepoints. We compared the change in those patterns against the change in experimental scaffold images seeded with C10 epithelial cells and mesenchymal stem cells, two cell types whose behaviors are not well understood, in order to determine which rulesets more closely match each cell type. Our model indicates that C10s are more likely to survive on regions of higher substrate while MSCs are more likely to proliferate on regions of higher substrate

Poster Presentations from the 2018 Maine Medical Center Research Institute (MMCRI) Summer Student Research Program

The following posters were presented as part of the 2018 MMCRI Summer Student Research Program. This program offers undergraduates and medical students a unique opportunity to conduct research in diverse clinical and biomedical science fields during the summer months. During the paid ten-week program, students participate in mentored independent research projects either in our state-of-the-art research facility, or working with physicians in a hospital setting to impact patient care or the outcome of treatment. Students also attend lectures and workshops featuring topics including bioethics, animal use in biomedical science and scientific presentation skills, and have the opportunity to attend presentations by guest scientists and MMCRI faculty. All students give a final presentation, which in 2018 involved a three minute oral presentation called a “Three Minute Thesis” as well as a scientific poster presentation. All authors have an affiliation with MMCRI, unless otherwise noted

Epigenetic Regulators as the Gatekeepers of Hematopoiesis

Hematopoiesis is the process by which both fetal and adult organisms derive the full repertoire of blood cells from a single multipotent progenitor cell type, the hematopoietic stem cells (HSCs). Correct enactment of this process relies on a synergistic interplay between genetically encoded differentiation programs and a host of cell-intrinsic and cell-extrinsic factors. These include the influence of the HSC niche microenvironment, action of specific transcription factors, and alterations in intracellular metabolic state. The consolidation of these inputs with the genetically encoded program into a coherent differentiation program for each lineage is thought to rely on epigenetic modifiers. Recent work has delineated the precise contributions of different classes of epigenetic modifiers to HSC self-renewal as well as lineage specification and differentiation into various cell types. Here, we bring together what is currently known about chromatin status and the development of cells in the hematopoietic system under normal and abnormal conditions

Hematopoiesis and T-cell specification as a model developmental system

The pathway to generate T cells from hematopoietic stem cells guides progenitors through a succession of fate choices while balancing differentiation progression against proliferation, stage to stage. Many elements of the regulatory system that controls this process are known, but the requirement for multiple, functionally distinct transcription factors needs clarification in terms of gene network architecture. Here, we compare the features of the T-cell specification system with the rule sets underlying two other influential types of gene network models: first, the combinatorial, hierarchical regulatory systems that generate the orderly, synchronized increases in complexity in most invertebrate embryos; second, the dueling ‘master regulator’ systems that are commonly used to explain bistability in microbial systems and in many fate choices in terminal differentiation. The T-cell specification process shares certain features with each of these prevalent models but differs from both of them in central respects. The T-cell system is highly combinatorial but also highly dose-sensitive in its use of crucial regulatory factors. The roles of these factors are not always T-lineage-specific, but they balance and modulate each other's activities long before any mutually exclusive silencing occurs. T-cell specification may provide a new hybrid model for gene networks in vertebrate developmental systems

Implications of metabolism-driven myeloid dysfunctions in cancer therapy

Immune homeostasis is maintained by an adequate balance of myeloid and lymphoid responses. In chronic inflammatory states, including cancer, this balance is lost due to dramatic expansion of myeloid progenitors that fail to mature to functional inflammatory neutrophils, macrophages, and dendritic cells (DCs), thus giving rise to a decline in the antitumor effector lymphoid response. Cancer-related inflammation orchestrates the production of hematopoietic growth factors and cytokines that perpetuate recruitment and activation of myeloid precursors, resulting in unresolved and chronic inflammation. This pathologic inflammation creates profound alterations in the intrinsic cellular metabolism of the myeloid progenitor pool, which is amplified by competition for essential nutrients and by hypoxia-induced metabolic rewiring at the tumor site. Therefore, persistent myelopoiesis and metabolic dysfunctions contribute to the development of cancer, as well as to the severity of a broad range of diseases, including metabolic syndrome and autoimmune and infectious diseases. The aims of this review are to (1) define the metabolic networks implicated in aberrant myelopoiesis observed in cancer patients, (2) discuss the mechanisms underlying these clinical manifestations and the impact of metabolic perturbations on clinical outcomes, and (3) explore new biomarkers and therapeutic strategies to restore immunometabolism and differentiation of myeloid cells towards an effector phenotype to increase host antitumor immunity. We propose that the profound metabolic alterations and associated transcriptional changes triggered by chronic and overactivated immune responses in myeloid cells represent critical factors influencing the balance between therapeutic efficacy and immune-related adverse effects (irAEs) for current therapeutic strategies, including immune checkpoint inhibitor (ICI) therapy

The Transcriptional Landscape of Hematopoietic Stem and Progenitor Cells during Acute Inflammatory Stress

Hematopoietic stem cells (HSCs) are critical components of the hematopoietic system and are responsible for renewing all blood cell lineages throughout life. These cells are quiescent and reside in niches in the bone marrow (BM). Over the past decade, our group and others have discovered that inflammatory stress impacts quiescent HSCs in vivo, leading to their activation. However, the dynamics, heterogeneity, and mechanisms underlying stress-induced activation of HSCs remain unclear. In this thesis, I unraveled the mechanisms regulating HSCs proliferation and recovery in response to acute treatment with the proinflammatory cytokine interferon alpha(IFNα) by initially determining three-time points representing the sensing, proliferation, and recovery phases of HSCs' proliferative response to acute IFNα treatment. Using time series bulk RNA sequencing (RNAseq), I identified distinct molecular patterns and changes in the activation and repression of various biological categories in HSCs. Surprisingly, even after returning to a quiescent state 72 hours (h) post-treatment, HSCs remained metabolically active and underwent a significant metabolic shift towards oxidative phosphorylation (OXPHOS). In addition, the tricarboxylic acid cycle (TCA), pentose phosphate pathway (PPP), fatty acid, and purine metabolism were reduced, and HSCs showed decreased myeloid priming and bias. Thus far, little is known about the dynamics and heterogeneity of these stress responses in the whole hematopoietic stem and progenitor (HSPC) cells. Inflammation-induced marker changes in the HSPCs compartment make it challenging to investigate the heterogeneity in the inflammatory response in HSPCs. Thus, I employed a single-cell (Sc) time series RNAseq experiment to study the heterogeneous and dynamic impacts of IFNα on HSPCs. The results showed heterogeneity in the response of HSPCs to IFNα, with HSCs being the strongest responders based on their gene expression changes. In collaboration with Brigitte Bouman and Dr. Laleh Haghverdi at the MDC in Berlin, we developed and used a response-pseudotime inference approach to analyze the scRNAseq data and identified global and cell type-specific inflammation signatures, revealing unique molecular patterns of gene expression and biological processes in response to IFNα. Interestingly, we were able to associate reduced myeloid differentiation programs in HSPCs with a reduced abundance of myeloid progenitors and differentiated cells following IFNα treatment. Taken together, the single-cell time series analyses have allowed us to unbiasedly study the heterogeneous and dynamic impact of IFNα on the HSPCs. In addition to investigating the dynamics and heterogeneity of the response of HSCs to IFNα, I compared the immediate transcriptional response of HSCs to various other proinflammatory cytokines. This analysis showed that IFNs, TNFα, ILs, and mimetics of viral and bacterial infections induced unique gene alterations in HSCs, underscoring the diversity of cytokine responses in these cells. Finally, I investigated how the baseline levels of these proinflammatory cytokines regulate hematopoiesis. Analysis of the hematopoietic system in Ifnar-/-Ifngr-/- (2KO) and Ifnar-/-Ifngr-/-Tnfrsf1dKOIl1r-/- (5KO) mice under homeostatic conditions revealed a decrease in HSCs and LSKs compared with wild-type (WT) mice. Furthermore, HSCs from these cytokine receptors knockout (KO) mice showed impaired colony-forming capacity and early competitive advantage. Interestingly, 5KO mice also showed a delayed recovery of HSCs cycling following 5-FU treatment. In addition, bulk RNA sequencing of 5KO HSCs revealed altered cell cycle pathways. Overall, these results underscore the essential role of proinflammatory cytokines in regulating HSC function during homeostasis. In conclusion, this thesis comprehensively explains the transcriptional changes within the HSPCs population in response to proinflammatory cytokines, focusing on IFNα

Aging and Health

Aging is a major risk factor for chronic diseases, which in turn can provide information about the aging of a biological system. This publication serves as an introduction to systems biology and its application to biological aging. Key pathways and processes that impinge on aging are reviewed, and how they contribute to health and disease during aging is discussed. The evolution of this situation is analyzed, and the consequences for the study of genetic effects on aging are presented. Epigenetic programming of aging, as a continuation of development, creates an interface between the genome and the environment. New research into the gut microbiome describes how this interface may operate in practice with marked consequences for a variety of disorders. This analysis is bolstered by a view of the aging organism as a whole, with conclusions about the mechanisms underlying resilience of the organism to change, and is expanded with a discussion of circadian rhythms in aging

Proceedings of the 96th Annual Virginia Academy of Science Meeting, 2018

Proceedings of the 96th Annual Virginia Academy of Science Meeting, May 23-25, 2018, at Longwood University, Farmville, Virginia

Spatial and temporal features of neutrophils in homeostasis from the perspective of computational biology

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 22-07-2022Neutrophils are myeloid cells that originate in the Bone Marrow and enter circulation to patrol for infectious agents. An important part of the “nonspecific” immune system consists on Neutrophils infiltrating challenged tissues, and the established belief was that they stay away from steady-state organs to avoid the risk of exposing them to their cytotoxic content. In the papers presented in this thesis, we show that neutrophils can in fact be found in almost all tissues under homeostasis. We further present proof that they undergo shifts in DNA accessibility, RNA expression and protein content in the infiltrated tissues. Using functional annotation we predict distinct roles depending on the tissue. While in hematopoietic organs the transcriptomic signatures of neutrophils align with canonical functions like immune response and migration, in other tissues such as the skin we find non-canonical functions i.e, epithelial and connective tissue growth or pro-angiogenic roles in the gut and the lung. This predicted pro-angiogenic role was indeed confirmed for the lung. We finally describe that infiltration in tissues follows circadian dynamics, and that once it has occurred, neutrophils experience changes in transcription depending on the time of the day. The analyses of circadian rhythms on mammalian models are often hindered by the inherent difficulty of performing exhaustive sampling (i.e.: every hour for at least 48h). Hence, I implemented CircaN as an R package, which outperforms existing tools in most scenarios. To provide the most complete analysis possible, we provide a full mode analysis option, in which we run CircaN and the two most used algorithms and provide integrated results. We present proof-of-concept results showing that combining various tools yields the best true positive to false positive ratio for most purposesEsta Tesis ha sido financiada por el Ministerio de Ciencia, Innovación y Universidades (MICINN