Single‐cell RNA Sequencing Identified Novel Nr4a1+ Ear2+ Anti‐Inflammatory Macrophage Phenotype under Myeloid‐TLR4 Dependent Regulation in Anti‐Glomerular Basement Membrane (GBM) Crescentic Glomerulonephritis (cGN)

Abstract

Abstract Previously, this study demonstrates the critical role of myeloid specific TLR4 in macrophage‐mediated progressive renal injury in anti‐glomerular basement membrane (anti‐GBM) crescentic glomerulonephritis (cGN); however, the underlying mechanism remains largely unknown. In this study, single‐cell RNA sequencing (scRNA‐seq), pseudotime trajectories reconstruction, and motif enrichment analysis are used, and macrophage diversity in anti‐GBM cGN under tight regulation of myeloid‐TLR4 is uncovered. Most significantly, a myeloid‐TLR4 deletion‐induced novel reparative macrophage phenotype (Nr4a1+Ear2+) with significant upregulated anti‐inflammatory and tissue repair‐related signaling is discovered, thereby suppressing the M1 proinflammatory responses in anti‐GBM cGN. This is further demonstrated in vitro that deletion of TLR4 from bone marrow‐derived macrophages (BMDMs) induces the Nr4a1/Ear2‐expressing anti‐inflammatory macrophages while blocking LPS‐stimulated M1 proinflammatory responses. Mechanistically, activation of the Nr4a1/Ear2‐axis is recognized as a key mechanism through which deletion of myeloid‐TLR4 promotes the anti‐inflammatory macrophage differentiation in vivo and in vitro. This is confirmed by specifically silencing macrophage Nr4a1 or Ear2 to reverse the anti‐inflammatory effects on TLR4 deficient BMDMs upon LPS stimulation. In conclusion, the findings decode a previously unidentified role for a myeloid‐TLR4 dependent Nr4a1/Ear2 negative feedback mechanism in macrophage‐mediated progressive renal injury, implying that activation of Nr4a1‐Ear2 axis can be a novel and effective immunotherapy for anti‐GBM cGN.

Keywords

• anti‐GBM crescentic glomerulonephritis
• macrophages
• myeloid‐TLR4
• Nr4a1/Ear2