The Structure and Function of Dental Lymphatics (R21)

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The lymphatic vessel (LV) system is a parallel circulatory system to the blood circulatory system present in almost all tissues. One major function of the LV system is the return of extravasated interstitial fluid to the blood vessel (BV) circulation. In addition, LV act as conduits for the traffic of antigens and antigen-presenting cells from peripheral tissue sites to draining lymph nodes, thereby permitting the induction of adaptive immunity. The existence of LV in dental pulp is still a controversial issue; no consensus as to whether LV are present in the dental pulp has hitherto been reached. In addition, even in those studies that support the existence of dental LV, the overall organization of the dental LV system and its three-dimensional (3D) structure has not been demonstrated. Furthermore, whether a putative dental LV system is involved in immune-inflammatory regulation, similar to the role of LV in other organ systems, is an important question that has not been addressed. We have recently identified a 3D network of LV in dental pulp using a Prox1-eGFP mouse model in which the Prox1 promoter drives expression of eGFP specifically in lymphatic endothelial cells (LEC). As imaged by Two-photon microscopy, the network comprises of microcapillaries (≃3-5 um in diameter) that form dense plex- uses mainly in the pulp chamber and which connect to larger vessels (≃10-15 um in diameter) that travel as a bundle from the pulp chamber floor through the root canals. In addition, we found that infection of dental pulp with Fusobacterium nucleatum resulted in disappearance of coronal microcapillary LV plexuses within 48 hours, suggesting that pulpal infection induces rapid death of LEC in the dental pulp. The structural and functional relationship of components of the identified dental LV network to each other and to the different types of LV that have been identified in other organs is unknown. In addition, the mechanism of infection-induced LV loss has not been determined. Answers to these questions are expected to inform upon the role of dental LV in host immunity to dental pathogens. In this proposal, the assembled multidisciplinary team will further investigate the structure, function, and pathophysiological changes of the dental pulp LV network using an innovative animal model, two-photon micros- copy, a dental infection model, and a fluorescent particle-tracking assay to confirm that the identified LV network conveys pulpal material to draining lymph nodes. There are two specific aims as follows: Aim 1. To characterize normal lymphatics in mouse dental pulp Aim 2. To examine the response of dental pulp lymphatics to pulpal infection Successful completion of the proposed aims will significantly advance our understanding of LV biology, pulp biology, and the immune response to dental infection and is expected to lead to follow-up projects that focus upon host pathogen relationships in the dental pulp.