Simon J. Cleary

University of California, San Francisco

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Mark R. Looney

University of California, San Francisco

Mélia Magnen

Université de Tours

Simon C. Pitchford

King's College London

Clive P. Page

King's College London

Longhui Qiu

University of California, San Francisco

Richard T. Amison

King's College London

Catharina Conrad

Columbia University Irving Medical Center

Yurim Seo

University of California, San Francisco

Jonathan P. Singer

University of California, San Francisco

Jennifer J. Tian

Columbia University Irving Medical Center

Cooperative Institutions

University of California, San Francisco

149

King's College London

University of California System

University of Cambridge

Advanced ENT and Allergy

UCSF Helen Diller Family Comprehensive Cancer Center

University of Washington

Zhejiang University

Wellcome/MRC Cambridge Stem Cell Institute

University of Rochester

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Intravital imaging of pulmonary lymphatics in inflammation and metastatic cancer

The Journal of Experimental Medicine (2025)

Simon J. Cleary Longhui Qiu Yurim Seo Peter Bałuk Dan Liu

Intravital microscopy has enabled the study of immune dynamics in the pulmonary microvasculature, but many key events remain unseen because they occur in deeper lung regions. We therefore developed a technique for stabilized intravital imaging of bronchovascular cuffs and collecting lymphatics surrounding pulmonary veins in mice. Intravital imaging of pulmonary lymphatics revealed ventilation dependence of steady-state lung lymph flow and ventilation-independent lymph flow during inflammation. We imaged the rapid exodus of migratory dendritic cells through lung lymphatics following inflammation and measured effects of pharmacologic and genetic interventions targeting chemokine signaling. Intravital imaging also captured lymphatic immune surveillance of lung-metastatic cancers and lymphatic metastasis of cancer cells. To our knowledge, this is the first imaging of lymph flow and leukocyte migration through intact pulmonary lymphatics. This approach will enable studies of protective and maladaptive processes unfolding within the lungs and in other previously inaccessible locations.

Intravital microscopy

10.1084/jem.20241359

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Functional haematopoietic progenitor cells in the adult human lung

Research Square (Research Square) (2023)

Catharina Conrad Mélia Magnen Jessica Tsui Harrison Wismer Mohammad Naser

Abstract The bone marrow is classically viewed as the main site of blood cell production in adults, however, rare pools of haematopoietic stem and progenitor cells with self-renewal and differentiation potential have been described in extramedullary organs. The lung is primarily known for its role in gas exchange but has recently been identified as a site of blood (platelet) production in mice. Here, we show that functional haematopoietic precursors reside in the extravascular spaces of the human lung, at a frequency similar to the bone marrow, and are capable of proliferation and engraftment. Comparative transcriptomic profiling of pulmonary and medullary CD34 + haematopoietic progenitor cells revealed organ-specific gene signatures and indicated greater baseline activation of immune, megakaryocyte/platelet and erythroid-related pathways in progenitors from the lung. In the lung, these cells predominately reside in the vascular-rich alveolar interstitium and near macrophages. These results identify the lung as an important niche for uniquely programmed blood stem and progenitor cells with considerable potential to support haematopoiesis in humans.

Progenitor

10.21203/rs.3.rs-3576483/v1

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Gustav Born: pioneer in imaging platelet and leukocyte biology

Platelets (2018)

Simon J. Cleary Clive P. Page

Gustav Born achieved scientific fame for his application of light transmission aggregometry to the study of platelet function, but also led interdisciplinary research teams in pioneering quantitative in vivo imaging studies of platelet aggregation and leukocyte adhesion, and in conducting the first research into the biomechanical factors underlying atherosclerotic plaque rupture. Gus Born also communicated both current research findings and an integrated understanding of cardiovascular biology to a wide audience through acting as scientific advisor on several television productions. Using footage from two of these films, we discuss Gustav Born’s scientific achievements and legacy.

10.1080/09537104.2018.1535001

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Optimizing anesthesia and delivery approaches for dosing into lungs of mice

AJP Lung Cellular and Molecular Physiology (2023)

Yurim Seo Longhui Qiu Mélia Magnen Catharina Conrad S. Farshid Moussavi-Harami

Many lung research studies involve dosing fluids into lungs of mice. In this study, the authors measure lung deposition using intranasal (i.n.), oropharyngeal aspiration (o.a.), and intratracheal (i.t.) dosing methods in mice. Anesthetic approach and administration route were found to affect pulmonary dosing efficiency. The authors demonstrate that refinements to dosing techniques can enable reductions in the number of animals needed for bacterial and viral pneumonia studies.

10.1152/ajplung.00046.2023

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Natural killer cells activated through NKG2D mediate lung ischemia-reperfusion injury

Journal of Clinical Investigation (2020)

Daniel R. Calabrese Emily Aminian Beñat Mallavia Fengchun Liu Simon J. Cleary

Pulmonary ischemia-reperfusion injury (IRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. IRI causes early mortality and has no effective therapies. While NK cells are innate lymphocytes capable of recognizing injured cells, their roles in acute lung injury are incompletely understood. Here, we demonstrated that NK cells were increased in frequency and cytotoxicity in 2 different IRI mouse models. We showed that NK cells trafficked to the lung tissue from peripheral reservoirs and were more mature within lung tissue. Acute lung ischemia-reperfusion injury was blunted in a NK cell-deficient mouse strain but restored with adoptive transfer of NK cells. Mechanistically, NK cell NKG2D receptor ligands were induced on lung endothelial and epithelial cells following IRI, and antibody-mediated NK cell depletion or NKG2D stress receptor blockade abrogated acute lung injury. In human lung tissue, NK cells were increased at sites of ischemia-reperfusion injury and activated NK cells were increased in prospectively collected human bronchoalveolar lavage in subjects with severe IRI. These data support a causal role for recipient peripheral NK cells in pulmonary IRI via NK cell NKG2D receptor ligation. Therapies targeting NK cells may hold promise in acute lung injury.

NKG2D

10.1172/jci137047

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In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET

ACS Central Science (2021)

Ning Zhao Conner Bardine André Luiz Lourenço Yung-Hua Wang Yangjie Huang

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Granzyme

Proteolysis

Granzyme A

10.1021/acscentsci.1c00529

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Faculty Opinions recommendation of Dipeptidase-1 Is an Adhesion Receptor for Neutrophil Recruitment in Lungs and Liver.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature (2019)

Mark R. Looney Simon J. Cleary

10.3410/f.736478100.793566660

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P-Rex and Vav Rac-GEFs in platelets control leukocyte recruitment to sites of inflammation

Blood (2014)

Dingxin Pan Richard T. Amison Yanira Riffo‐Vasquez Domenico Spina Simon J. Cleary

10.1182/blood-2014-07-591040

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Citations (76)

Lung megakaryocytes are immune modulatory cells

Journal of Clinical Investigation (2020)

Daphne N. Pariser Zachary T. Hilt Sara Ture Sara K. Blick-Nitko Mark R. Looney

Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.

10.1172/jci137377

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Optimizing anesthesia and delivery approaches for dosing into lungs of mice

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Yurim Seo Longhui Qiu Mélia Magnen Catharina Conrad S. Farshid Moussavi-Harami

Microbes, toxins, therapeutics and cells are often instilled into lungs of mice to model diseases and test experimental interventions. Consistent pulmonary delivery is critical for experimental power and reproducibility, but we observed variation in outcomes between handlers using different anesthetic approaches for intranasal dosing into mice. We therefore used a radiotracer to quantify lung delivery after intranasal dosing under inhalational (isoflurane) versus injectable (ketamine/xylazine) anesthesia in C57BL/6 mice. We found that ketamine/xylazine anesthesia resulted in delivery of a greater proportion (52±9%) of an intranasal dose to lungs relative to isoflurane anesthesia (30±15%). This difference in pulmonary dose delivery altered key outcomes in models of viral and bacterial pneumonia, with mice anesthetized with ketamine/xylazine for intranasal infection with influenza A virus or Pseudomonas aeruginosa developing more robust lung inflammation responses relative to control animals randomized to isoflurane anesthesia. Pulmonary dosing efficiency through oropharyngeal aspiration was not affected by anesthetic method and resulted in delivery of 63±8% of dose to lungs, and a non-surgical intratracheal dosing approach further increased lung delivery to 92±6% of dose. Use of either of these more precise dosing methods yielded greater experimental power in the bacterial pneumonia model relative to intranasal infection. Both anesthetic approach and dosing route can impact pulmonary dosing efficiency. These factors affect experimental power and so should be considered when planning and reporting studies involving delivery of fluids to lungs of mice.

10.1101/2023.02.01.526706

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