Project Grant F31HL189412

SPATIAL IDENTITY OF LUNG LYMPHATICS AND THE ROLE OF PKHD1L1 IN DEVELOPMENT AND PNEUMONIA REMODELING - PROJECT ABSTRACT PULMONARY LYMPHATICS ARE ESSENTIAL FOR DRAINING INTERSTITIAL FLUID FROM THE LUNGS AND TRAFFICKING IMMUNE CELLS FROM THE LUNGS TO LYMPH NODES, ESPECIALLY DURING LUNG DEVELOPMENT AND PNEUMONIA WHICH NATURALLY CAUSE EDEMA. HOWEVER, THERE REMAINS A SIGNIFICANT KNOWLEDGE GAP IN PULMONARY LYMPHATIC FORMATION AND REMODELING DURING PNEUMONIA. THIS PROPOSAL AIMS TO ADDRESS THE KNOWLEDGE GAP BY FURTHER UNDERSTANDING THE ROLE OF LYMPHATIC ENDOTHELIAL CELLS (LECS), WHICH LINE THE PULMONARY LYMPHATIC VESSELS. OUR LAB IDENTIFIED TWO CLUSTERS OF LECS WITH DISTINCT TRANSCRIPTOMES AND SPATIAL ORGANIZATION USING SINGLE NUCLEI RNA SEQUENCING ON ISOLATED LECS FROM THE HOMEOSTATIC MOUSE LUNG: (1) LECS NEAR PULMONARY VEINS AND DISTAL AIRSPACES (PVLECS) AND (2) LECS NEAR BRONCHOVASCULAR BUNDLES WITH PULMONARY ARTERIES AND AIRWAYS (BVLECS). BVLECS UNIQUELY EXPRESS POLYCYSTIC KIDNEY AND HEPATIC DISEASE 1-LIKE 1 (PKHD1L1), WHICH WAS ORIGINALLY STUDIED FOR STEREOCILIA BUNDLING IN HEARING LOSS. BASED ON OUR PRELIMINARY DATA, PKHD1L1 EXPRESSION IS BARELY PRESENT DURING MOUSE EMBRYONIC LUNG DEVELOPMENT, THEN IS QUICKLY INDUCED DURING NEONATAL DEVELOPMENT. DURING BACTERIAL PNEUMONIA, PKHD1L1 EXPRESSION IS UPREGULATED IN MICE THAT HAVE DEVELOPED HETEROTYPIC IMMUNITY BY SUSTAINING REPEATED PNEUMOCOCCAL INFECTIONS. DISTINCT INCREASES OF PKHD1L1 INDICATE PKHD1L1 MAY HAVE A FUNCTIONAL ROLE IN REGULATING PULMONARY LYMPHATIC GROWTH, DILATION, FLUID CLEARANCE, AND IMMUNE CELL TRAFFICKING IN THE BVLEC NICHE. TAKING THIS TOGETHER, WE HYPOTHESIZE THAT PULMONARY LYMPHATIC HETEROGENEITY ESTABLISHES SPECIALIZED VASCULAR CIRCUITS IN THE LUNGS, AND PKHD1L1 BRONCHOVASCULAR LECS REPRESENT AN ADAPTIVE SUBSET THAT ENABLES POSTNATAL LYMPHATIC MATURATION, VESSEL DILATION, AND IMMUNE-PROTECTIVE REMODELING DURING HETEROTYPIC IMMUNITY. WE WILL USE A TWO-AIM APPROACH TO ADDRESS OUR HYPOTHESIS. AIM 1 WILL CREATE A BROAD UNDERSTANDING OF PULMONARY LEC TRANSCRIPTOMIC HETEROGENEITY IN CONNECTION WITH NICHE-SPECIFIC REMODELING BY USING SPATIAL TRANSCRIPTOMICS IN MOUSE DEVELOPING LUNGS AND LUNGS WITH HETEROTYPIC IMMUNITY. ADDITIONALLY, WE WILL CHARACTERIZE EMERGENCE OF PVLECS AND BVLECS DURING DEVELOPMENT ALONG WITH LYMPHANGIOGENESIS, VESSEL DILATION, AND IMMUNE CELL MOVEMENT IN BVLEC NICHES VERSUS PVLEC NICHES DURING HETEROTYPIC IMMUNITY. AIM 2 WILL UTILIZE A TAMOXIFEN INDUCIBLE PROX1-CRE-ERT2/PKHD1L1FL/FL MOUSE MODEL FOR LEC-SPECIFIC PKHD1L1 DELETION TO IDENTIFY IF PKHD1L1 REGULATES BVLEC DEVELOPMENT AFTER BIRTH AND WHETHER PKHD1L1 RELATED BVLEC REMODELING CONTRIBUTES TO THE PROTECTIVE PHENOTYPE IN HETEROTYPIC IMMUNITY. THESE STUDIES WILL EMPLOY HISTOLOGY AND FLOW CYTOMETRY TECHNIQUES AND WILL BE PERFORMED WITHIN THE COLLABORATIVE, SHARED LABORATORY OF THE PULMONARY CENTER AND CORE FACILITIES. AS A TRAINEE, THE PROPOSED STUDIES WILL HONE MY CRITICAL THINKING ABOUT SCIENTIFIC AND EXPERIMENTAL RATIONALE, AND DEVELOP MY EXPERTISE IN BIOINFORMATICS, PULMONARY LYMPHATIC BIOLOGY, AND THE ART OF COMMUNICATING MY RESEARCH FINDINGS TO HELP ME BECOME AN INDEPENDENT INVESTIGATOR.