Pulmonary lymphatic remodeling in response to influenza-induced inflammation
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Abstract
The lymphatic system consists of a vessel network lined by specialized lymphatic endothelial cells (LECs) that are responsible for tissue fluid homeostasis and immune cell trafficking. LECs also promote regeneration through lymphangiocrine signaling and perform immunomodulatory functions. The mechanisms for organ-specific LEC responses to environmental cues are still not well understood. We aimed to characterize how pulmonary lymphatics change during influenza and identify mechanisms that drive their responses to this disease. We found robust lymphangiogenesis during influenza A virus infection in the adult mouse lung. We show that the number of LECs increases 2-fold at 7 days post-influenza infection (dpi) and 3-fold at 21 dpi, and that lymphangiogenesis is preceded by lymphatic dilation. We also show that the expanded lymphatic network enhances fluid drainage to mediastinal lymph nodes. Using EdU labeling, we found that a significantly higher number of pulmonary LECs are proliferating at 7 dpi compared to LECs in homeostatic conditions. Lineage tracing during influenza indicates that new pulmonary LECs are derived from preexisting LECs rather than non-LEC progenitors. As the Hippo pathway effectors YAP and TAZ play a key role in developmental lymphangiogenesis, we next asked how their activity affects LEC responses during influenza. Using a conditional LEC-specific YAP/TAZ knockout model, we established that lymphangiogenesis, fluid transport and the immune response to influenza are independent of YAP/TAZ activity in LECs. These findings were unexpected, as they indicate that YAP/TAZ signaling is not crucial for these processes.We also asked whether subsets of pulmonary LECs with distinct phenotypes and functions exist beyond the broad categories of initial lymphatics, pre-collectors, collectors and valves. To begin to address this knowledge gap, we performed two transcriptomic studies using single nuclei RNA sequencing (snRNAseq). In one study, we focused on LECs at baseline. In the other, we compared the transcriptomes of LECs from influenza-infected lungs to those from mock-infected controls. LECs make up a small proportion of lung endothelial cells, so in order to obtain a larger and more representative subset of the target population for sequencing, we tested several enrichment methods. This was technically challenging due to LEC rarity and fragility. We had some success with fluorescence-activated nuclear sorting (FANS) of GFP-tagged LEC nuclei for snRNAseq, although numbers of sorted nuclei were lower than anticipated. We found that during homeostasis, LECs clustered into two groups with subtle transcriptional differences mainly linked to cell shape and interactions with the extracellular matrix (ECM). Pathway analysis showed that these distinctions were mainly linked to cell shape and interactions with extracellular matrix.
Attempts to enrich for LECs from influenza-infected lungs and controls also yielded a low number of target nuclei, but snRNAseq did show significant differential expression of various genes involved in the LEC response to the disease. The transcriptomes of LEC nuclei from influenza-infected lungs showed upregulation of many pathways involved in the antiviral response, such as the interferon-beta, gamma and alpha responses. Pathways involved with cell-matrix adhesion and morphogenesis were downregulated in influenza. The data generated through both of these sequencing experiments is a foundation for future research. In addition to validating findings from these transcriptomic studies using techniques such as RNAScope, further investigation will include inhibiting lymphangiogenesis via inducible VEGFR3 knockout in LECs prior to influenza infection. By employing this strategy, we aim to further elucidate the role of the expanded lymphatic vasculature in immunomodulatory functions, lymphangiocrine signaling and fluid transport. A better understanding of these responses to influenza will clarify the effect of lymphatic function and dysfunction on both tissue resilience and viral clearance.
Description
2024