Role of lymphatic endothelial cell NFkB signaling in adult lymph node homeostasis

PROJECT SUMMARY/ABSTRACT The proposed study is significant because we aim to understand the cellular mechanisms that regulate lymph node (LN) maintenance in aging. Aging causes LN atrophy and fibrosis and is associated with impaired immune responses. However, although the morphological changes that occur in aged LNs have been well

studied, the molecular mechanisms that cause these changes are poorly understood. This gap in knowledge is important because LNs play a key role in orchestrating immune responses. Therefore, our studies will help uncover therapies that may improve immune function in elderly men and women. Lymphatic endothelial cells (LECs) are the building blocks of the lymphatic system and, as a network of

lymphatic vessels, transport of interstitial fluid and antigens to regional LNs. LECs are also a component of the LN stroma and regulate LN organogenesis during development and LN hypertrophy in response to inflammatory conditions. These regulatory pathways are coordinated by canonical and non-canonical nuclear factor kappa-

light-chain-enhancer of activated B cells (NFkB) signaling in LECs. Aging is known to impair lymphatic function and transport of interstitial fluid/antigens to the LN, and we have shown in preliminary studies that LN LEC heterogeneity is altered in aged LNs and the expression of NFkB by LN stromal LEC is decreased in aging animals. We also have found that deleting canonical NFkB-Rel-A

signaling in LECRel-A-/- mice results in significant LN atrophy and cellularity that is a phenocopy of the aging LN. Based on this rationale, our central hypothesis is that decreased lymphatic function in aging results in diminished expression of NFkB by LN stromal LECs, impaired production of lymphocyte survival signals, and

aging-related LN atrophy. Our study is innovative because we plan to conduct single-cell RNA sequencing analysis of LN stromal LECs, and we have developed inducible transgenic Cre-lox mice in which we can selectively decrease LEC NFκB signaling in adult animals. This model system enables us to directly study the role of LEC NFκB expression

in LN maintenance and atrophy. These transgenic mice, combined with our LN transplantation, and in vitro 3D models, will also enable us to selectively analyze changes in LN LEC NFκB signaling and understand how these populations contribute to adult LN homeostasis using two Specific Aims: (1) Determine the effects of aging on

LN LECs and effect of lymphatic flow on LEC NFkB expression and (2) determine how LEC NFκB signaling regulates LN homeostasis in adult mice. At the conclusion of the proposed study, we expect to understand the cellular mechanisms that contribute to aging-related LN atrophy and adult LN homeostasis. This understanding will provide the basis for future

studies designed to prevent or reverse aging-related LN atrophy.