Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models
Alison C. Livada, Kathleen E. McGrath, Michael W. Malloy, Chen Li, Sara K. Ture, Paul D. Kingsley, Anne D. Koniski, Leah A. Vit, Katherine E. Nolan, Deanne Mickelsen, Grace E. Monette, Preeti Maurya, James Palis, Craig N. Morrell
Alison C. Livada, Kathleen E. McGrath, Michael W. Malloy, Chen Li, Sara K. Ture, Paul D. Kingsley, Anne D. Koniski, Leah A. Vit, Katherine E. Nolan, Deanne Mickelsen, Grace E. Monette, Preeti Maurya, James Palis, Craig N. Morrell
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Research Article Hematology

Long-lived lung megakaryocytes contribute to platelet recovery in thrombocytopenia models

Abstract

Lung megakaryocytes (Mks) are largely extravascular with an immune phenotype (1). Because bone marrow (BM) Mks are short lived, it has been assumed that extravascular lung Mks are constantly “seeded” from the BM. To investigate lung Mk origins and how origin affects their functions, we developed methods to specifically label lung Mks using CFSE dye and biotin delivered via the oropharyngeal route. Labeled lung Mks were present for up to 4 months, while BM Mks had a lifespan of less than 1 week. In a parabiosis model, lung Mks were partially replaced over 1 month from a circulating source. Unlike tissue-resident macrophages, using MDS1-Cre-ERT2 TdTomato mice, we found that lung Mks arose from hematopoietic stem cells. However, studies with FlkSwitch mTmG mice showed that lung Mks were derived from a Flt3-independent lineage that did not go through a multipotent progenitor. CFSE labeling to track lung Mk–derived platelets showed that approximately 10% of circulating platelets were derived from lung-resident Mks at steady state, but in sterile thrombocytopenia this was doubled (~20%). Lung-derived platelets were similarly increased in a malaria infection model (Plasmodium yoelii) typified by thrombocytopenia. These studies indicate that lung Mks arise from a Flt3– BM source, are long-lived, and contribute more platelets during thrombocytopenia.

Authors

Alison C. Livada, Kathleen E. McGrath, Michael W. Malloy, Chen Li, Sara K. Ture, Paul D. Kingsley, Anne D. Koniski, Leah A. Vit, Katherine E. Nolan, Deanne Mickelsen, Grace E. Monette, Preeti Maurya, James Palis, Craig N. Morrell

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Figure 1

Lung Mks are long-lived cells.

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Lung Mks are long-lived cells. (A and B) CFSE or (C) biotin was delivere...
(A and B) CFSE or (C) biotin was delivered o.p. to mice and showed specific labeling of lung Mks, but not BM or splenic Mks. (A) CFSE 6 hours later (n = 4, 2 independent experiments), (B) CFSE on day 1 (d1) to day 7 (d7) (n = 4–5 per group from 2 independent experiments) and (C) on day 5 after biotin o.p. labeling (n = 4; results are representative of 2 independent experiments). (D) CFSE-labeled lung Mks were present up to 120 days after CFSE administration (n = 10 from 2 independent experiments for day 30; n = 4 for day 120 from 1 independent experiment). L, lung; Spl, spleen. (E) Representative flow cytometry and quantitation results 6 hours after biotin i.v. labeling of BM, splenic, and lung Mks, similar to A (n = 4; results are representative of 2 independent experiments). (F) Biotin-labeled lung but not BM or splenic Mks were present up to 28 days following i.v. delivery (n = 4; results are representative of 2 independent experiments). RBCs were used as a positive control. PB, peripheral blood. (G) Total lung Mks and CFSE+ Mks had similar ploidy (day 14 after CFSE) (n = 5, representative results are from 2 independent experiments). (H) CFSE– and CFSE+ lung Mks had a similar intravascular (CD42d+) and extravascular (CD42d–) distribution (CD42d given i.v.) (n = 5; results are representative of 2 independent experiments). Data indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by (B) multiple t tests with Holm-Šidák multiple-comparison correction, (D and F) 2-way ANOVA with Tukey’s multiple-comparison correction, and (H) 1-way ANOVA with Šidák multiple-comparison correction. Ctl, control; FSC, forward scatter.