Development of a new macrophage-specific TRAP mouse (MacTRAP) and definition of the renal macrophage translational signature

Abstract

Tissue macrophages play an important role in organ homeostasis, immunity and the pathogenesis of various inflammation-driven diseases. One major challenge has been to selectively study resident macrophages in highly heterogeneous organs such as kidney. To address this problem, we adopted a Translational Ribosome Affinity Purification (TRAP)-approach and designed a transgene that expresses an eGFP-tagged ribosomal protein (L10a) under the control of the macrophage-specific c-fms promoter to generate c-fms-eGFP-L10a transgenic mice (Mac TRAP). Rigorous characterization found no gross abnormalities in Mac TRAP mice and confirmed transgene expression across various organs. Immunohistological analyses of Mac TRAP kidneys identified eGFP-L10a expressing cells in the tubulointerstitial compartment which stained positive for macrophage marker F4/80. Inflammatory challenge led to robust eGFP-L10a upregulation in kidney, confirming Mac TRAP responsiveness in vivo. We successfully extracted macrophage-specific polysomal RNA from Mac TRAP kidneys and conducted RNA sequencing followed by bioinformatical analyses, hereby establishing a comprehensive and unique in vivo gene expression and pathway signature of resident renal macrophages. In summary, we created, validated and applied a new, responsive macrophage-specific TRAP mouse line, defining the translational profile of renal macrophages and dendritic cells. This new tool may be of great value for the study of macrophage biology in different organs and various models of injury and disease.

Network analysis of transcriptomic diversity amongst resident tissue macrophages and dendritic cells in the mouse mononuclear phagocyte system

“…However, our analysis also revealed 2 important artefacts in the study of isolated tissue macrophages: the clear evidence of inflammatory activation during isolation and the extensive contamination of isolated preparations with transcripts derived from other cell types. One recent innovation that may obviate these issues is the use of a so-called MacTrap transgene to isolated macrophage-specific polysome-associate mRNA from tissues without isolating the cells [ 193 ].…”

Profile of Podocyte Translatome During Development of Type 2 and Type 1 Diabetic Nephropathy Using Podocyte-Specific TRAP mRNA RNA-seq

“…As a key regulatory node in gene expression, the profile of mRNA undergoing translation correlates better with actual protein expression than total mRNA and is more relevant to the cell phenotype (16,17). To date, the use of the TRAP approach in kidney cells has only been reported in acute kidney injury, focal segmental glomerulosclerosis and fibrosis, and unilateral ureteral obstruction (18)(19)(20)(21). For better understanding of the role of podocytes in DN, both type 1 and type 2 diabetic mouse models were used to investigate the translating mRNA profiling of podocytes with use of the TRAP approach.…”

Distinct Hypoxia-induced Translational Profiles of Embryonic and Adult-derived Macrophages

“…The attenuation of miRNA binding to transcripts in close proximity to HuR binding sites, and consequential miRNA targeting of transcripts regulating angiogenesis and macrophage/endothelial interactions, have been demonstrated (Chang et al, 2013). There are global and specific changes in macrophage mRNA translation in response to various stimuli, including hypoxia (Hofmeister et al, 2020; Liu et al, 2014; Rahat, 2011; Shibata et al, 2013). However, prior studies have either focused on bone marrow-derived macrophages (BMDM) or simply did not attempt to address the origin of macrophages.…”