A molecular map of murine lymph node blood vascular endothelium at single cell resolution

Blood vascular endothelial cells (BECs) control the immune response by regulating blood flow and immune cell recruitment in lymphoid tissues. However, the diversity of BEC and their origins during immune angiogenesis remain unclear. Here we profile transcriptomes of BEC from peripheral lymph nodes and map phenotypes to the vasculature. We identify multiple subsets, including a medullary venous population whose gene signature predicts a selective role in myeloid cell (vs lymphocyte) recruitment to the medulla, confirmed by videomicroscopy. We define five capillary subsets, including a capillary resident precursor (CRP) that displays stem cell and migratory gene signatures, and contributes to homeostatic BEC turnover and to neogenesis of high endothelium after immunization. Cell alignments show retention of developmental programs along trajectories from CRP to mature venous and arterial populations. Our single cell atlas provides a molecular roadmap of the lymph node blood vasculature and defines subset specialization for leukocyte recruitment and vascular homeostasis

Recruitment of neutrophils to the lymph node and their role at steady state and after Staphylococcus aureus infection

The contribution of lymph nodes to the adaptive immune response is well studied. However, their function as an innate immune organ has been overlooked. In this thesis, neutrophils are identified in the lymph node at steady state. The entry of these neutrophils is entirely dependent upon L-selectin. After entering the lymph node, neutrophils at steady state will recirculate, exiting from the lymph node via efferent lymphatics in an S1P-dependent manner. In mice co-housed with pet store mice, there is an increased number of neutrophils in the lymph nodes at steady state. These neutrophils act as reconnaissance cells to recruit additional neutrophils after infection. We observed that after infection in the foot, there is very little dissemination past the popliteal lymph node and into the peripheral organs. In four hours, the number of neutrophils in the lymph node increases dramatically from steady state. Neutrophil entry proceeds through the high endothelial venules (HEV) and can occur via direct L-selectin-Peripheral node addressin (PNAd) interactions or through a bridging mechanism involving neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) and platelet- P-selectin binding to PNAd on HEV. C5a is necessary for neutrophil recruitment, but it is independent of C-C chemokine receptor type 7 (CCR7) and lymph node resident macrophages. Blocking neutrophil recruitment using anti-PNAd antibody resulted in bacterial dissemination, demonstrating the contribution of neutrophils to the innate immune function of the lymph node. Finally, this thesis begins to assess the contribution of lymph node innervation to the above processes. Sympathetic innervation may play a greater role than sensory innervation, at least as it pertains to neutrophils, as determined by the methods used here. This work still establishes an important basis for future examination of the effect of innervation on other cell types in the lymph node. Overall, this body of work has made important contributions to the understanding of neutrophil biology in the context of the lymph node, both at steady state and after infection

Incorporating Immune Cells into Organoid Models: Essential for Studying Human Disease

Organoid-based research has made significant discoveries and contributions to our understanding of human organ function in both health and disease. To continue making progress, it is crucial to acknowledge the crucial role of the immune system in all organs. Various immune cells, such as macrophages, T cells, and neutrophils, are resident in almost all human tissues and play essential roles in organ homeostasis, function, and disease. Using diverse methods, researchers have begun integrating immune cells into organoid models, leading to more physiologically relevant models that better represent various aspects of human disease. These methods range from immune cell injection to co-culture and tissue expansion with existing immune cells. Immune cells can be sourced from mature patients or generated from stem cells as immature immune cells. The successful incorporation of immune cells into organoids will enhance our understanding of organ function and provide a more accurate approximation of human disease.Medicine, Faculty ofNon UBCMedical Genetics, Department ofReviewedFacultyResearche

Neutrophils recirculate through lymph nodes to survey tissues for pathogens

The adaptive immune function of lymph nodes is dependent on constant recirculation of lymphocytes. In this article, we identify neutrophils present in the lymph node at steady state, exhibiting the same capacity for recirculation. In germ-free mice, neutrophils still recirculate through lymph nodes, and in mice cohoused with wild microbiome mice, the level of neutrophils in lymph nodes increases significantly. We found that at steady state, neutrophils enter the lymph node entirely via L-selectin and actively exit via efferent lymphatics via an S1P dependent mechanism. The small population of neutrophils in the lymph node can act as reconnaissance cells to recruit additional neutrophils in the event of bacterial dissemination to the lymph node. Without these reconnaissance cells, there is a delay in neutrophil recruitment to the lymph node and a reduction in swarm formation following Staphylococcus aureus infection. This ability to recruit additional neutrophils by lymph node neutrophils is initiated by LTB4. This study establishes the capacity of neutrophils to recirculate, much like lymphocytes via L-selectin and high endothelial venules in lymph nodes and demonstrates how the presence of neutrophils at steady state fortifies the lymph node in case of an infection disseminating through lymphatics