Marzullo, B. | Search bioRxiv

bioRxiv2021

Transcriptome profiling reveals CD73 and age-driven changes in neutrophil responses against Streptococcus pneumoniae

Neutrophils are required for host resistance against Streptococcus pneumoniae but their function declines with age. We previously found that CD73, an enzyme required for antimicrobial activity, is down-regulated in neutrophils from aged mice. This study explored transcriptional changes in neutrophils induced by S. pneumoniae to identify pathways controlled by CD73 and dysregulated with age. Ultrapure bone marrow-derived neutrophils isolated from wild type (WT) young, old, and CD73KO young mice were mock-challenged or infected with S. pneumoniae ex vivo. RNA sequencing was performed to identify differentially expressed genes (DEGs). We found that infection triggered distinct global transcriptional changes across hosts, that were strongest in CD73KO neutrophils. Surprisingly, there were more down-regulated than up-regulated genes in all groups upon infection. Down-regulated DEGs indicated a dampening of immune responses in old and CD73KO hosts. Further analysis revealed that CD73KO neutrophils expressed higher numbers of long non-coding RNAs (lncRNAs) compared to WT controls. Predicted network analysis indicated that CD73KO specific lncRNAs control several signaling pathways. We found that genes in the JNK-MAPK-pathway were up-regulated upon infection in CD73KO and WT old but not in young mice. This corresponded to functional differences, as phosphorylation of the downstream AP-1 transcription factor component c-Jun was significantly higher in infected CD73KO and old mice neutrophils. Importantly, inhibiting JNK/AP-1 rescued the ability of these neutrophils to kill S. pneumoniae. Altogether, our findings revealed that neutrophils modify their gene expression to better adapt to bacterial infection and that this capacity declines with age and is regulated by CD73.

immunology↗

bioRxiv2020

Neuromesodermal Progenitors Advance Network Formation of Spinal Neurons and Support Cells in Neural Ribbons In Vitro and Unprotected Survival in a Rat Subacute Contusion Model

Improved human stem cell interventions to treat CNS trauma requires continued expansion of in vitro models and delivery platforms to fill gaps in analysis and treatment. Transplanted neural stem cells (NSCs) face unique, multi-faceted challenges beyond survival that include differentiation, maturation, and integration into a complex cytokine-releasing microenvironment that impinges on a multipotent cell type. Alternate strategies to transplant neurons and neuronal networks deserve reevaluation, particularly since novel differentiation protocols mimicking region-specific developmental and positional cues have recently emerged. To investigate transplantation of neurons and their early networks, we generate in vitro neural ribbons containing spinal neurons and support cells anatomically matched for cervical spinal cord injury (SCI). These glutamate-responsive, electrically-active neural ribbons apply a new hiPSC differentiation strategy transiting through neuromesodermal progenitors (NMps) to derive developmentally relevant spinal motor neurons (SMNs), interneurons (INs), and oligodendrocyte progenitor cells (OPCs). Bioinformatic profiling validates region-specific identities. Neurons and neuronal networks are functionally evaluated for action potential firing, calcium signaling, population activity, and synaptogenesis. NMp-derived neurons survive in vivo within the subacute phase hemi-contusion injury cavity when delivered either as free suspension or as encapsulated networks of pre-formed CNS cytoarchitectures. Delivery as encapsulated networks further supports survival of lower cell numbers and rapid graft penetration into host tissue. Neural network ribbons therefore provide a novel intermediary approach between cell suspensions and complex organoids for investigating network formation and early transplantation events with hiPSC-derived neurons, providing flexibility to rapidly tune cell type(s), cell ratios, and traceable biomarkers. Significance StatementIn the two decades since human stem cell technologies have emerged, the challenge has remained to improve the developmentally relevant derivation of therapeutic cells. The ability to now generate anatomically matched neurons for SCI necessitates a re-evaluation of these cells and their networks in vitro and in vivo. In this study, we apply developmental cues via neuromesodermal progenitors to generate spinal neurons from hiPSCs. Genetic and functional evaluation of these cells as in vitro neuronal networks, due to their capacity to survive and graft effectively within the rat subacute contusion cavity, offer novel approaches for customizing SCI transplantation. This work demonstrates a strategy to develop transplantable, chemically-responsive networks linking in vitro models with injury customization towards improved in vivo outcomes.

neuroscience↗