Lung megakaryocytes display distinct transcriptional and phenotypic properties

Abstract

Megakaryocytes (MKs) are responsible for platelet biogenesis, which is believed to occur canonically in adult bone marrow (BM) and in the fetal liver during development. However, emerging evidence highlights the lung as a previously underappreciated residence for MKs that may contribute significantly to circulating platelet mass. Although a diversity of cells specific to the BM is known to promote the maturation and trafficking of MKs, little investigation into the impact of the lung niche on the development and function of MKs has been done. Here, we describe the application of single-cell RNA sequencing, coupled with histological, ploidy, and flow cytometric analyses, to profile primary MKs derived from syngeneic mouse lung and hematopoietic tissues. Transcriptional profiling demonstrated that lung MKs have a unique signature distinct from their hematopoietic counterparts, with lung MKs displaying enrichment for maturation markers, potentially indicating a propensity for more efficient platelet production. Reciprocally, fetal lung MKs also showed the robust expression of cytokines and growth factors that are known to promote lung development. Lastly, lung MKs possess an enrichment profile skewed toward roles in immunity and inflammation. These findings highlight the existence of a lung-specific MK phenotype and support the notion that the lung plays an independent role in the development and functional maturation of MKs. The immune phenotype displayed by lung MKs also introduces their potential role in microbial surveillance and antigen presentation.

Graphical abstract

Figure 1.

Adult lung MKs are localized to the alveolar interstitium of the distal lung, and fetal lung MKs are found intra- and extravascularly. (A-C) Immunofluorescent staining of 5- to 6-µm fixed-frozen week-10 (WK10) adult mouse lung sections stained for markers of MKs (CD41a and CD42d), lung epithelial cells (NKX2.1), alveolar type 1 epithelial cells (PDPN), and nuclei (Hoechst). Lung borders and an alveolar airspace are outlined with gray dashes. Multinucleated adult lung MKs were preferentially localized to the interstitium of the alveolar septum between neighboring alveoli. (D-E) Immunofluorescent staining of 5- to 6-µm fixed-frozen E13 fetal mouse lung sections stained for VE-Cadherin (endothelial cell marker), CD41a, NKX2.1, and Hoechst. Fetal lung MKs were identified in the developing lung in extravascular (D) and intravascular (E) spaces. Platelets were notably less prevalent in the fetal lung. White and red arrows highlight platelets and MKs, respectively.

Figure 2.

Lung MKs are transcriptionally distinct from their hematopoietic counterparts. (A,C) UMAP clustering of MK-lineage clusters from fetal liver (FLiv-1, FLiv-2), fetal lung (Flu), adult BM (ABM-1, ABM-2, ABM-3), and ALu. Fetal and adult lung MKs clustered independently from their hematopoietic counterparts. Numbers in parentheses indicate cell counts for associated clusters. (B,D) Violin plots comparing expression of key MK genes in associated clusters. Key genes used to confirm MK lineage identity are outlined with a dashed box. Colors of violin plots in panels B and D correlate with their respective clusters shown in panels A and C, respectively. *P < .01. (Asterisks indicating significance were withheld from panel B for visual clarity.)

Figure 3.

Fetal lung MKs display transcriptional priming for platelet and cytokine production. (A) SPRING plot of fetal MK clusters. Directionality of maturation progresses from left to right and was determined based on key markers, as described in supplemental Figure 4. Percentages indicate which fraction of the outlined cells is represented by each respective cluster. (B) Gene set enrichment analysis of the corresponding clusters that are outlined in a dashed box in panel A. (C) Violin plots comparing the expression of various cytokines and growth factors across all fetal MK clusters. *P < .0001.

Figure 4.

Adult lung MKs exhibit higher ploidy and express markers of maturation. (A) Ploidy analysis of CD41a⁺ MKs demonstrated a greater proportion of high ploidy MKs from the lung. (B-C) Flow cytometric assessment showed that a greater proportion of lung MKs was positive for the key maturation marker CD42d, which was significant across 3 biological replicates. BSA enrichment was used for these experiments. Ploidy analysis without BSA enrichment was also performed and showed similar results (supplemental Figure 5). "X" and "●" represent individual data points, whereas the horizontal bars are averages. *P < .05.

Figure 5.

Differential gene-expression and pathway analysis demonstrate the immune phenotype of fetal lung MKs. Heat map and gene set enrichment analysis illustrating robust upregulation of immune/inflammatory genes in fetal lung MKs. The top 20 differentially expressed genes associated with each cluster are listed to the left of the heat map. The gene ontology pathways associated with the remaining clusters are shown in supplemental Figure 6.

Figure 6.

Differential gene expression and pathway analysis demonstrate the immune phenotype of adult lung MKs. Heat map and gene set enrichment analysis illustrating robust upregulation of immune/inflammatory genes in adult lung MKs. Adult BM cluster 1 showed enrichment for processes important in proplatelet production, and this gene set was notably also upregulated in the adult lung population (dashed black box). The top 20 differentially expressed genes associated with each cluster are listed to the left of the heat map. The gene ontology pathways associated with the remaining clusters are shown in supplemental Figure 7.

Figure 7.

Adult Lung MKs exhibit significant upregulation of immune markers. (A) Gene-expression analysis of various TLRs, chemokines, and the surface immune marker Cd74 showed a distinct expression pattern with markedly more enrichment for many of these genes in lung MKs. (B) Flow cytometric assessment of TLR2, TLR4, CD74, and MHCII was performed on CD41a⁺/CD42d⁺ MKs. (C) Percentage of cells expressing these immune markers was quantified across 3 replicates, which demonstrated significantly higher expression of TLR2, TLR4, and MHCII in lung MKs. BSA enrichment was not used prior to immune marker analyses. *P < .05.