Loss of transcription factor myb drives an immune differentiated megakaryocyte phenotype Free

Bulk and single cell RNA-sequencing (RNA-seq) of lung and bone marrow (BM) megakaryocytes (Mks) revealed the transcription factor Myb was expressed in BM but not lung Mks. These RNA-seq results were verified by qPCR and suggested transcriptomic heterogeneity between lung and BM Mks. Here, we report Mk Myb expression changes in response to immune and inflammatory stimuli, the functional consequences of BM and lung Mk Myb expression, and a Myb-dependent platelet phenotype.

We measured Mk Myb expression following immune challenges by utilizing a GFP-Myb reporter mouse, in which a GFP locus under the Myb gene promoter is transcribed in all Myb-expressing cells. Using this mouse model, we determined that about 90% of BM Mks express Myb in basal conditions, while lung Mks had minimal GFP positivity. We found that Mk Myb expression is dynamic and responds to immune challenges and inflammatory cues. In an acute model of inflammation, GFP-Myb mice received daily intraperitoneal (IP) injections of 0.04 mg/kg interferon gamma (IFNγ) for 3 days. After 3 days of IFNγ treatment, the percentage of Myb positive BM Mks decreased to about 75%. To model a chronic pathological infection model with an IFNγ dominant response, we infected Myb-GFP mice with the non-lethal murine malaria strain Plasmodium Yoelii (PyNL). 14 days after PyNL infection, the percentage of Myb positive BM Mks decreased to 20%. This was validated by qPCR for Myb in Mks isolated from infected and healthy control mice. Furthermore, in both the models of acute and chronic inflammation, the Myb negative Mks present in the BM were phenotypically distinct from the Myb positive BM Mks, as indicated by increased expression of ICAM1 and MHCII.

To evaluate the role of Myb in regulating Mk phenotype, we generated a megakaryocyte and platelet specific Myb knockout mouse (PF4^cre x Myb^flx/flx, PF4-Myb). By comparing the BM and lung Mks from WT and PF4-Myb mice, we determined that BM Mks from PF4-Myb mice have a similar phenotype to WT lung Mks. Specifically, compared to WT BM Mks, PF4-Myb Mks have lower ploidy and increased expression of ICAM1, MHCII and TLR7 by flow cytometry, a phenotype similar to lung Mks.

To identify lung-derived platelets in circulation, we delivered the cell permeable dye CFSE to mice through the oropharyngeal (OP) route. Lung (CFSE+) and BM (CFSE-) platelets were isolated by flow sorting and compared by RNA-seq. RNA-seq revealed that lung-derived platelets have increased expression of Tlr7, similar to Myb negative Mks. We found that both lung-derived platelets and platelets from PF4-Myb mice have increased degranulation, measured by surface CD62p expression, in response to the TLR7 agonist Loxoribine. We also observed an increase in platelet-leukocyte aggregate (PLA) formation in response to Loxoribine by PF4-Myb platelets.

Previously reported as a regulator of erythroid lineage fate, here we present a novel role of the transcription factor Myb. Mk Myb expression is associated with the low ploidy immune Mk phenotype of lung Mks. BM Mks respond to infection and inflammation by switching to a Myb negative transcriptional program, in which immune proteins such as ICAM1, MHCII and TLR7 are upregulated. Functional consequences of a Mk phenotypic shift are reflected in platelets, as platelets from Myb negative Mks, either lung-derived or from PF4-Myb mice, have increased activation following Loxoribine stimulation and increased leukocyte aggregation.