Regulatory role and mechanisms of myeloid TLR4 in anti-GBM glomerulonephritis

Abstract

Myeloid cells and TLR4 play a critical role in acute kidney injury. This study investigated the regulatory role and mechanisms of myeloid TLR4 in experimental anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Anti-GBM GN was induced in tlr4flox/flox and tlr4flox/flox−lysM−cre mice by intravenous injection of the sheep anti-mouse GBM antibody. Compared to control mice, conditional disruption of tlr4 from myeloid cells, largely macrophages (> 85%), suppressed glomerular crescent formation and attenuated progressive renal injury by lowering serum creatinine and 24-h urine protein excretion while improving creatinine clearance. Mechanistically, deletion of myeloid tlr4 markedly inhibited renal infiltration of macrophages and T cells and resulted in a shift of infiltrating macrophages from F4/80+iNOS+ M1 to F4/80+CD206+ M2 phenotype and inhibited the upregulation of renal proinflammatory cytokines IL-1β and MCP-1. Importantly, deletion of myeloid tlr4 suppressed T cell-mediated immune injury by shifting Th1 (CD4+IFNγ+) and Th17 (CD4+IL-17a+) to Treg (CD4+CD25+FoxP3+) immune responses. Transcriptome analysis also revealed that disrupted myeloid TLR4 largely downregulated genes involving immune and cytokine-related pathways. Thus, myeloid TLR4 plays a pivotal role in anti-GBM GN by immunological switching from M1 to M2 and from Th1/Th17 to Treg and targeting myeloid TLR4 may be a novel therapeutic strategy for immune-mediated kidney diseases.

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)

“…[13][14][15] As a result, resident macrophage signatures, such as Mrc1(Cd206), Cd74, Cd81, C1qa, C1qc were highly expressed in Clusters 4, 8, and 9 macrophages (Figure 2D). Consistent with our previous findings, [11] deletion of myeloid TLR4 did not alter the proportion of the tissue resident macrophages (Cluster 4, 8, and 9) between Tlr4 flox/flox and Tlr4 fl/fl /LysM-cre anti-GBM cGN mice group (Figure 1D). On the other hand, recruited monocyte/macrophages, identified by expressing Cd14, Sell, Cxcr2, Ifitm1, were significantly upregulated in Cluster 0 and 1, which markedly demonstrating the M1 proinflammatory macrophage phenotype and were largely suppressed in the diseased kidney when myeloid-TLR4 was deleted (Figure 1D and 2D-F).…”

“…[11] As reported previously, compared to the Tlr4 flox/flox littermates, the majority of TLR4 (>85%) was successfully and effectively deleted from macrophages in Tlr4 fl/fl /LysM-cre mice, as assessed at mRNA level by real time PCR and at protein level by flow cytometry, both in BMDMs and in the kidney macrophages with anti-GBM cGN in Tlr4 fl/fl /LysM-cre mice. [11] All animals were raised under a specific pathogen-free condition at 25 °C with a normal 12-h light and 12-h dark cycle. Mice were allowed free access to standard food and sterilized water supplied by the animal unit in the Chinese University of Hong Kong.…”

“…It is highly possible that the Nr4a1/Ear2-expressing macrophages may have renoprotective effect in anti-GBM cGN as evidenced by deletion of TLR4 inhibits progressive renal functional and histological injury, including glomerular crescentic formation in Tlr4 fl/fl /LysMcre mice. [11] The discovery of a TLR4-dependent novel Nr4a1/Ear2 antiinflammatory macrophage population was the most important finding in this study. This was supported by the finding that deletion of myeloid-TLR4 induced a novel population of macrophages (89% derived from Tlr4 fl/fl /LysM-cre cells) uniquely expressing Nr4a1 and Ear2, which is transcriptomically distinguished from any known M1 and M2 phenotypes.…”

“…Mice with myeloid‐specific TLR4 deletion ( Tlr4 fl/fl /LysM‐cre ) were generated by crossing C57BL/6 mice bearing homozygous loxP ‐flanked Tlr4 ( Tlr4 flox/flox ) (JAX stock number: 024872) and C57BL/6 mice with lysozyme M promoter‐driven cre ( LysM‐cre ) (JAX stock number: 004781) as previously described. [ 11 ] As reported previously, compared to the Tlr4 flox/flox littermates, the majority of TLR4 (>85%) was successfully and effectively deleted from macrophages in Tlr4 fl/fl /LysM‐cre mice, as assessed at mRNA level by real time PCR and at protein level by flow cytometry, both in BMDMs and in the kidney macrophages with anti‐GBM cGN in Tlr4 fl/fl /LysM‐cre mice. [ 11 ]…”

“…Our previous study demonstrated that deletion of myeloid‐TLR4 markedly alleviated progressive cGN and largely improved renal function by suppressing macrophage and neutrophil infiltration, and promoting macrophage polarization into the anti‐inflammatory M2 phenotype. [ 11 ] However, transcriptional regulation through which deletion of myeloid TLR4 leads to the shift of macrophages from the M1 to M2 phenotype in anti‐GBM cGN remains to be elucidated.…”