Article Open Access

Single Cell Analysis of Lung Lymphatic Endothelial Cells and Lymphatic Responses during Influenza Infection

Journal of Respiratory Biology and Translational Medicine . 2024, 1(1), 10003;
Jian Ge 1    Hongxia Shao 1,2    Hongxu Ding 3    Yuefeng Huang 4    Xuebing Wu 5    Jie Sun 6    Jianwen Que 1 *   
Columbia Center for Human Development & Division of Digestive and Liver Diseases, Department of Medicine, Columbia University Irving Medical Center, New York, NY 10032, USA
Haihe Hospital, Tianjin University, Tianjin 300350, China
Department of Pharmacy Practice and Science, College of Pharmacy, University of Arizona, Tucson, AZ 85724 USA
Department of Microbiology & Immunology, Columbia University Irving Medical Center, New York, NY 10032, USA
Department of Medicine, Department of Systems Biology, Columbia University Irving Medical Center, New York, NY 10032, USA
Carter Immunology Center, University of Virginia, Charlottesville, VA 22908, USA
Authors to whom correspondence should be addressed.

Received: 05 Feb 2024    Accepted: 18 Feb 2024    Published: 19 Feb 2024   


Tissue lymphatic vessels network plays critical roles in immune surveillance and tissue homeostasis in response to pathogen invasion, but how lymphatic system per se is remolded during infection is less understood. Here, we observed that influenza infection induces a significant increase of lymphatic vessel numbers in the lung, accompanied with extensive proliferation of lymphatic endothelial cells (LECs). Single-cell RNA sequencing illustrated the heterogeneity of LECs, identifying a novel PD-L1+ subpopulation that is present during viral infection but not at steady state. Specific deletion of Pd-l1 in LECs elevated the expansion of lymphatic vessel numbers during viral infection. Together these findings elucidate a dramatic expansion of lung lymphatic network in response to viral infection, and reveal a PD-L1+ LEC subpopulation that potentially modulates lymphatic vessel remolding.


Donnan MD, Kenig-Kozlovsky Y, Quaggin SE. The lymphatics in kidney health and disease. Nat. Rev. Nephrol. 2021, 17, 655–675. [Google Scholar]
Oliver G, Kipnis J, Randolph GJ, Harvey NL. The Lymphatic Vasculature in the 21st Century: Novel Functional Roles in Homeostasis and Disease. Cell 2020, 182, 270–296. [Google Scholar]
Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat. Rev. Mol. Cell. Biol. 2007, 8, 464–478. [Google Scholar]
Morrow PE. Lymphatic drainage of the lung in dust clearance. Ann. N. Y. Acad. Sci. 1972, 200, 46–65. [Google Scholar]
Schraufnagel DE, Agaram NP, Faruqui A, Jain S, Jain L, Ridge KM, et al. Pulmonary lymphatics and edema accumulation after brief lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 2003, 284, L891–L897. [Google Scholar]
Jakus Z, Gleghorn JP, Enis DR, Sen A, Chia S, Liu X, et al. Lymphatic function is required prenatally for lung inflation at birth. J. Exp. Med. 2014, 211, 815–826. [Google Scholar]
Escobedo N, Oliver G. Lymphangiogenesis: Origin, Specification, and Cell Fate Determination. Ann. Rev. Cell Dev. Biol. 2016, 32, 677–691. [Google Scholar]
Srinivasan RS, Escobedo N, Yang Y, Interiano A, Dillard ME, Finkelstein D, et al. The Prox1-Vegfr3 feedback loop maintains the identity and the number of lymphatic endothelial cell progenitors. Genes Dev. 2014, 28, 2175–2187. [Google Scholar]
Wigle JT, Harvey N, Detmar M, Lagutina I, Grosveld G, Gunn MD, et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 2002, 21, 1505–1513. [Google Scholar]
Kumar PA, Hu Y, Yamamoto Y, Hoe NB, Wei TS, Mu D, et al. Distal airway stem cells yield alveoli in vitro and during lung regeneration following H1N1 influenza infection. Cell 2011, 147, 525–538. [Google Scholar]
Zacharias WJ, Frank DB, Zepp JA, Morley MP, Alkhaleel FA, Kong J, et al. Regeneration of the lung alveolus by an evolutionarily conserved epithelial progenitor. Nature 2018, 555, 251–255. [Google Scholar]
Katsura H, Kobayashi Y, Tata PR, Hogan BLM. IL-1 and TNFalpha Contribute to the Inflammatory Niche to Enhance Alveolar Regeneration. Stem Cell Rep. 2019, 12, 657–666. [Google Scholar]
Vaughan AE, Brumwell AN, Xi Y, Gotts JE, Brownfield DG, Treutlein B, et al. Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature 2015, 517, 621–625. [Google Scholar]
Nabhan AN, Brownfield DG, Harbury PB, Krasnow MA, Desai TJ. Single-cell Wnt signaling niches maintain stemness of alveolar type 2 cells. Science 2018, 359, 1118–1123. [Google Scholar]
Baluk P, Naikawadi RP, Kim S, Rodriguez F, Choi D, Hong YK, et al. Lymphatic Proliferation Ameliorates Pulmonary Fibrosis after Lung Injury. Am. J. Pathol. 2020, 190, 2355–2375. [Google Scholar]
Baluk P, Adams A, Phillips K, Feng J, Hong YK, Brown MB, et al. Preferential Lymphatic Growth in Bronchus-Associated Lymphoid Tissue in Sustained Lung Inflammation. Am. J. Pathol. 2014, 184, 1577–1592. [Google Scholar]
Kasmani MY, Topchyan P, Brown AK, Brown RJ, Wu X, Chen Y, et al. A spatial sequencing atlas of age-induced changes in the lung during influenza infection. Nat. Commun. 2023, 14, 6597. [Google Scholar]
Melms JC, Biermann J, Huang H, Wang Y, Nair A, Tagore S, et al. A molecular single-cell lung atlas of lethal COVID-19. Nature 2021, 595, 114–119. [Google Scholar]
Negretti NM, Plosa EJ, Benjamin JT, Schuler BA, Habermann AC, Jetter CS, et al. A single-cell atlas of mouse lung development. Development 2021, 148, dev199512. [Google Scholar]
Niethamer TK, Stabler CT, Leach JP, Zepp JA, Morley MP, Babu A, et al. Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury. eLife 2020, 9, 28. [Google Scholar]
Fu S, Wang Y, Bin E, Huang H, Wang F, Tang N. c-JUN-mediated transcriptional responses in lymphatic endothelial cells are required for lung fluid clearance at birth. Proc. Natl. Acad. Sci. USA 2023, 120, e2215449120. [Google Scholar]
Fang Y, Shao H, Wu Q, Wong NC, Tsong N, Sime PJ, et al. Epithelial Wntless regulates postnatal alveologenesis. Development 2022, 149, dev199505. [Google Scholar]
Huang H, Fang Y, Jiang M, Zhang Y, Biermann J, Melms JC, et al. Contribution of Trp63(CreERT2)-labeled cells to alveolar regeneration is independent of tuft cells. eLife 2022, 11, e78217. [Google Scholar]
Praefcke GJK. Regulation of innate immune functions by guanylate-binding proteins. Int. J. Med. Microbiol. 2018, 308, 237–245. [Google Scholar]
Butte MJ, Keir ME, Phamduy TB, Sharpe AH, Freeman GJ. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 2007, 27, 111–122. [Google Scholar]
Francisco LM, Sage PT, Sharpe AH. The PD-1 pathway in tolerance and autoimmunity. Immunol. Rev. 2010, 236, 219–242. [Google Scholar]
Watanabe Y, Kinoshita A, Yamada T, Ohta T, Kishino T, Matsumoto N, et al. A catalog of 106 single-nucleotide polymorphisms (SNPs) and 11 other types of variations in genes for transforming growth factor-beta1 (TGF-beta1) and its signaling pathway. J. Hum. Genet. 2002, 47, 478–483. [Google Scholar]
Bengtsson E, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg A. The leucine-rich repeat protein PRELP binds perlecan and collagens and may function as a basement membrane anchor. J. Biol. Chem. 2002, 277, 15061–15068. [Google Scholar]
Bengtsson E, Aspberg A, Heinegard D, Sommarin Y, Spillmann D. The amino-terminal part of PRELP binds to heparin and heparan sulfate. J. Biol. Chem. 2000, 275, 40695–40702. [Google Scholar]
Zhang B, Li KY, Chen HY, Pan SD, Jiang LC, Wu YP, et al. Spindle and kinetochore associated complex subunit 1 regulates the proliferation of oral adenosquamous carcinoma CAL-27 cells in vitro. Cancer Cell Int. 2013, 13, 83. [Google Scholar]
Wu Y, Wang A, Zhu B, Huang J, Lu E, Xu H, et al. KIF18B promotes tumor progression through activating the Wnt/beta-catenin pathway in cervical cancer. OncoTargets Ther. 2018, 11, 1707–1720. [Google Scholar]
McHugh T, Gluszek AA, Welburn JPI. Microtubule end tethering of a processive kinesin-8 motor Kif18b is required for spindle positioning. J. Cell Biol. 2018, 217, 2403–2416. [Google Scholar]
Podgrabinska S, Braun P, Velasco P, Kloos B, Pepper MS, Skobe M. Molecular characterization of lymphatic endothelial cells. Proc. Natl. Acad. Sci. USA 2002, 99, 16069–16074. [Google Scholar]
Yazdani S, Poosti F, Kramer AB, Mirkovic K, Kwakernaak AJ, Hovingh M, et al. Proteinuria triggers renal lymphangiogenesis prior to the development of interstitial fibrosis. PLoS ONE 2012, 7, e50209. [Google Scholar]
Hardavella G, Tzortzaki EG, Siozopoulou V, Galanis P, Vlachaki E, Avgousti M, et al. Lymphangiogenesis in COPD: another link in the pathogenesis of the disease. Respir. Med. 2012, 106, 687–693. [Google Scholar]
Lara AR, Cosgrove GP, Janssen WJ, Huie TJ, Burnham EL, Heinz DE, et al. Increased lymphatic vessel length is associated with the fibroblast reticulum and disease severity in usual interstitial pneumonia and nonspecific interstitial pneumonia. Chest 2012, 142, 1569–1576. [Google Scholar]
Palikuqi B, Rispal J, Reyes EA, Vaka D, Boffelli D, Klein O. Lymphangiocrine signals are required for proper intestinal repair after cytotoxic injury. Cell Stem Cell 2022, 29, 1262–1272.e5. [Google Scholar]
Freeman GJ, Long AJ, Iwai Y, Bourque K, Chernova T, Nishimura H, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 2000, 192, 1027–1034. [Google Scholar]
Dong H, Zhu G, Tamada K, Chen L. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat. Med. 1999, 5, 1365–1369. [Google Scholar]
Lucas ED, Finlon JM, Burchill MA, McCarthy MK, Morrison TE, Colpitts TM, et al. Type 1 IFN and PD-L1 Coordinate Lymphatic Endothelial Cell Expansion and Contraction during an Inflammatory Immune Response. J. Immunol. 2018, 201, 1735–1747. [Google Scholar]
Erickson JJ, Gilchuk P, Hastings AK, Tollefson SJ, Johnson M, Downing MB, et al. Viral acute lower respiratory infections impair CD8+ T cells through PD-1. J. Clin. Invest. 2012, 122, 2967–2982. [Google Scholar]
Dutta A, Huang CT, Lin CY, Chen TC, Lin YC, Chang CS, et al. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs. Sci. Rep. 2016, 6, 32973. [Google Scholar]
Rutigliano JA, Sharma S, Morris MY, Oguin TH, 3rd, McClaren JL, Doherty PC, et al. Highly pathological influenza A virus infection is associated with augmented expression of PD-1 by functionally compromised virus-specific CD8+ T cells. J. Virol. 2014, 88, 1636–1651. [Google Scholar]
Geng Y, Liu X, Liang J, Habiel DM, Kulur V, Coelho AL, et al. PD-L1 on invasive fibroblasts drives fibrosis in a humanized model of idiopathic pulmonary fibrosis. JCI Insight 2019, 4, e125326. [Google Scholar]
Piao W, Li L, Saxena V, Iyyathurai J, Lakhan R, Zhang Y, et al. PD-L1 signaling selectively regulates T cell lymphatic transendothelial migration. Nat. Commun. 2022, 13, 2176. [Google Scholar]
Gkountidi AO, Garnier L, Dubrot J, Angelillo J, Harle G, Brighouse D, et al. MHC Class II Antigen Presentation by Lymphatic Endothelial Cells in Tumors Promotes Intratumoral Regulatory T cell-Suppressive Functions. Cancer Immunol. Res. 2021, 9, 748–764. [Google Scholar]
Cousin N, Cap S, Dihr M, Tacconi C, Detmar M, Dieterich LC. Lymphatic PD-L1 Expression Restricts Tumor-Specific CD8(+) T-cell Responses. Cancer Res. 2021, 81, 4133–4144. [Google Scholar]
Creative Commons

© 2024 by the authors; licensee SCIEPublish, SCISCAN co. Ltd. This article is an open access article distributed under the CC BY license (