Myeloid EGFR deficiency accelerates recovery from AKI via macrophage efferocytosis and neutrophil apoptosis

Abstract

Altered expression and activation of Epidermal Growth Factor Receptor (EGFR) is implicated in acute and chronic kidney injury. One of the important cellular sources of EGFR is the myeloid compartment, which plays roles in both acute kidney injury and subsequent fibrosis. Here we show in a murine ischemic acute kidney injury (AKI) model that myeloid deletion of EGFR promotes a pro-resolving, anti-inflammatory phenotype and increased efferocytotic capacity in macrophages. This leads to accelerated recovery in response to AKI and inhibited subsequent development of tubulointerstitial fibrosis. We find that selective EGFR deletion in neutrophils also accelerates recovery from ischemic kidney injury and reduces subsequent fibrosis. EGFR activation plays an essential role in increasing the life span of neutrophils in the injured kidney. Deletion of EGFR expression either in all murine myeloid cells or selectively in neutrophils decreases kidney neutrophil Mcl-1 expression and promotes neutrophil apoptosis, which is accompanied by accelerated recovery from organ injury and reduced subsequent fibrosis. These studies thus identify coordinated and complementary roles for EGFR activation in neutrophils and macrophages to exacerbate kidney injury.

Fig. 1. Selective deletion of EGFR in myeloid cells led to accelerated functional recovery after ischemic kidney injury and less post-ischemia renal fibrosis.

A Schematic of experimental protocol with IRI-UNX, indicating unilateral pedicle clamping for 28.5 min plus uninephrectomy. B Myeloid EGFR^−/− had accelerated functional recovery from the ischemic insult, indicated by more rapid BUN decline (n = 6, 7 and 8). C Myeloid EGFR^−/− mouse kidneys had less tubular injury 3 days after injury (n = 6). (D&E) Myeloid EGFR^−/− mice had decreased macrophage density, indicated by: D lower mRNA levels of F4/80 (n = 4 and 5) and E quantitative colocalization of CD68 and arginase 1 (ARG1) (n = 4), F Myeloid EGFR^−/− mice had lower kidney proinflammatory cytokines/chemokines, including Tnf, Il1a, Il1b, Il6, Ccl2, Ccl3 and Il23a (n = 4 and 5). G–I Myeloid EGFR^−/− mice had less kidney fibrosis, indicated by: G lower mRNA (n = 4 and 5) and H protein levels (n = 3 and 4) of profibrotic and fibrotic genes and I less Picrosirius red staining (n = 6) 4 weeks after initial ischemic injury. Scale bar = 50 μm for all. Data are means ± SD (B) or means ± SEM (C–G), *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2-way ANOVA followed by Tukey’s post hoc test for (B); 2 tailed Student’s t test (C–I).

Fig. 2. Myeloid EGFR deficiency led to increased resident macrophage subclusters but relative decreases in infiltrating macrophages.

A Both male EGFR^f/f (WT) and CD11b-Cre: EGFR^f/f (myeloid EGFR^−/−) mice underwent ischemic injury. The animals were sacrificed at days 1 and 3, and kidney leukocytes were enriched with CD45 microbeads for single cell RNA-seq (scRNA-seq). B scRNA-seq analysis identified clusters of kidney cells, and canonical markers of kidney cell populations were used to identify major cell types in the kidney: macrophages (Cd68, C1qb), neutrophils (S100a9, S100a8), B lymph cells (Cd79b), T lymph cells (Cd3e), NK cells (Nkg7), cDC1(Cd68, C1qb, Xcr1), cDC2 (Cd68, C1qb, Cd209a), and pDC (Siglech). C Egfr mRNA was mainly detected in myeloid neutrophils and macrophages in WT mice. D Eleven subclusters were identified corresponding to macrophages: Mac1-11. E Violin plot of representative genes in each subcluster. C1qc^High HP^low Mac: Mac1, 2, 4, 6, 8, 9, and 10, which tended to be resident macrophages; C1qc^low HP^high Mac: Mac3, 5, 7, and 11, which tended to be infiltration macrophages; proliferating macrophages (Mac 6), proinflammatory macrophages (Mac7 and Mac11) and pro-fibrotic macrophages (Mac 5, 7, and 11). F Gene Ontology (GO) analysis suggested that Mac1 and 2 clusters were associated with myeloid cell differentiation and small GTPase mediated signal transduction, Mac10 cluster with energy metabolism and Mac 9 cluster with regulation of apoptotic signaling pathways. G, H The percentage of cells in each cluster. Compared to WT mice, myeloid EGFR^−/− mice had significantly increased Mac1, 2, 6, 8, 9 and 10 but not Mac 3, 5, 7, and 11 subclusters. I Flow cytometry confirmed that 3 days after ischemic injury myeloid EGFR^−/− mouse kidneys had increased C1q^highHp^low resident macrophages with less apoptosis, while the C1q^lowHp^high infiltrating macrophages were decreased and had increased apoptosis compared to WT mice (n = 9 and 10). Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test.

Fig. 3. Myeloid EGFR deletion had less kidney neutrophil infiltration after ischemic injury.

Both male EGFR^f/f (WT) and CD11b-Cre: EGFR^f/f (myeloid EGFR^−/−) mice underwent ischemic injury. Kidney myeloid cells were evaluated with flow cytometry at different time points. A Renal CD45⁺ leukocytes, B CD45⁺CD11b⁺F4/80⁺ macrophages and C CD45⁺CD11b⁺Gr-1⁺ neutrophils were comparable between WT mice and myeloid EGFR^−/− mice at baseline and 2 h after ischemic injury, but both CD45⁺ leukocytes and neutrophils were lower in myeloid EGFR^−/− mice than WT mice at 16 and 72 h after ischemic injury while macrophages increased to similar amounts (n = 5, 6 and 7). D Nine clusters were identified corresponding to neutrophils: Neu1-9. Bubble plots showed representative genes for each subcluster. E The percentage of cells in each cluster. Compared to WT mice, myeloid EGFR deletion reduced Neu1 and Neu3 clusters but increased Neu2 cluster. F Gene Ontology (GO) Biological Process analysis indicated that Neu1 cluster was associated with neutrophil migration, chemotaxis and activation, Neu2 cluster with small GTPase mediated signal transduction and immune cell-cell reaction, and Neu3 cluster with TNF, IL17 and NF-κB signaling pathway activation. Data are means ± SD, *P < 0.05, ***P < 0.001, analyzed using 2-way ANOVA followed by Tukey’s post hoc test for (A–C).

Fig. 4. Mice with selective EGFR deletion in neutrophils had accelerated functional recovery associated with fewer renal neutrophils after ischemic injury.

A Schematic of the experimental protocol with IRI-UNX induced by unilateral pedicle clamping for 28.5 min plus uninephrectomy. B–D NeutEGFR^−/− mice had accelerated functional recovery from the ischemic insult, indicated by more rapid BUN decline (n = 4–8) (B), lower KIM1 and NGAL protein levels (n = 5) (C) at day 1 after ischemic injury. D Three days after ischemic injury, NeutEGFR^−/− mice had fewer renal CD45+ leukocytes and CD45⁺CD11b⁺Gr-1⁺ while CD45⁺CD11b⁺F4/80⁺ macrophage numbers were numerically but not significantly different than WT mice (n = 4 and 7). E–G: Immunofluorescent staining indicated: E, F increased numbers of p-EGFR expressing neutrophils (n = 6) in WT mice after ischemic injury and (E, G) significantly fewer p-EGFR+ neutrophils in NeutEGFR^−/− mice at 16 and 72 h after ischemic injury (n = 6). Scale bar = 50 μm. Data are means ± SD (B, F), means ± SEM (C, D, G), *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2-way ANOVA followed by Tukey’s post hoc test for B and F; 2 tailed Student’s t test for (C, D and G).

Fig. 5. Selective EGFR deletion in neutrophils attenuated development of kidney fibrosis after ischemia injury.

A Schematic of experimental protocol. B–D, G NeutEGFR^−/− mouse kidney had less injury, indicated by: B lower mRNA levels of Havcr1/Kim-1 (n = 6 and 8) and less fibrosis, indicated by: C lower protein (n = 5) and D mRNA levels (n = 6 and 8) of profibrotic and fibrotic components and G decreased quantitative Picrosirius red staining (n = 6). NeutEGFR^−/− mice had less kidney immune cell infiltration, indicated by: E lower mRNA levels of Emr1/F4/80, Cd68 and Ly6g/Gr1 (n = 6 and 8), F macrophage and neutrophil numbers (n = 6 and 7), and G quantitative F4/80 and Gr-1 staining (n = 6). Scale bar = 50 μm. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test.

Fig. 6. EGFR deletion in myeloid cells increased kidney macrophage efferocytosis ability.

Kidney macrophages were isolated with F4/80 microbeads from myeloid EGFR^−/− mice and WT mice 2 days after bilateral 35-min renal pedicle clamping. Bone marrow neutrophils were isolated from WT mice with Ly6G microbeads. Myeloid EGFR^−/− mouse kidney macrophages had increased ability to clear apoptotic neutrophils as indicated by: A Ex vivo pHrodo Red real-time efferocytosis assay with apoptotic bone marrow neutrophils (n = 6). B The full gating strategies for the flow cytometry to evaluate kidney macrophage efferocytosis. The effector kidney macrophages were isolated from WT, myeloid EGFR^−/− and NeutEGFR^−/− mice 2 days after ischemic injury using anti-F4/80 microbeads and stained with CytoTell^TM Blue dye. The bait bone marrow neutrophils were isolated from WT mice using anti-ly6G microbeads and stained with CFSE dye and apoptosis was induced by staurosporine. The effector cells alone and bait cells alone or a mixture of effectors and baits at a ratio of 1:4 were incubated overnight before processing for flow cytometry. For flow cytometry analysis strategy, first small debris were removed, and singlets selected. Cells were gated on CytoTell^TMblue positivity and CFSE positivity with macrophages only in the reaction or neutrophils only in reaction as controls. When both CFSE-labeled apoptotic neutrophils and CytoTell^TMblue-labeled macrophages were both in the reaction, CFSE and CytoTell^TMblue double positivity represented macrophages with engulfed apoptotic neutrophils (expressed as percentage of total events with event counts in parenthesis). C EGFR deletion in myeloid cells increased kidney macrophage efferocytosis ability (n = 4 and 6). Data are means ± SEM, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test.

Fig. 7. Selective neutrophil EGFR deletion increased neutrophil apoptosis.

A Gating strategy for flow cytometry analysis of cleaved caspase 3 positive apoptotic neutrophils in kidney. B WT, myeloid EGFR^−/−, and NeutEGFR^−/− mice underwent IRI-UNX for 16 h with or without treatment with emricasan, a pan-caspase inhibitor. Flow cytometry analysis of cleaved caspase 3 indicated that the percentage of kidney apoptotic neutrophils was significantly higher in both myeloid EGFR^−/− mice (n = 4 and 5) and NeutEGFR^−/− mice (n = 5 and 6) compared to their corresponding WT mice (n = 4 and 5). Emricasan treatment led to an increased number of kidney neutrophils due to inhibition of neutrophil apoptosis in both myeloid EGFR^−/− and NeutEGFR^−/− mice. C Cleaved caspase-9 and S100A9 co-staining indicated that emricasan treatment led to increased numbers of kidney neutrophils and decreased percentage of neutrophil apoptosis in NeutEGFR^−/− mice. (n = 4 and 6). Scale bar = 50 μm for all. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2-way ANOVA followed by Bonferroni’s post hoc test for (B), and 2 tailed Student’s t test for (C).

Fig. 8. EGFR signaling promoted renal neutrophil survival via maintaining antiapoptotic protein Mcl-1 levels after ischemic injury.

NeutEGFR^−/− and WT mice underwent ischemic injury and were sacrificed at the indicated time points. A Three days after ischemic injury, expression of both p-EGFR and Mcl-1 in kidney neutrophils was evident in WT mice but was minimal in NeutEGFR^−/− mice. B–D After ischemic injury, the number of Mcl-1 expressing neutrophils was comparable between WT and NeutEGFR^−/− mice at 2 h and 6 h but was markedly lower at 16 and 72 h in NeutEGFR^−/− mice (n = 5–8). E In isolated WT bone marrow neutrophils EGF administration for 16 h induced immunoreactive Mcl-1 protein, which was markedly attenuated in EGFR^−/− bone marrow neutrophils isolated from myeloid EGFR^−/− mice. Scale bar = 50 μm for all. Data are means ± SD, **P < 0.01, ***P < 0.001, analyzed using 2-way ANOVA followed by Tukey’s post hoc test.

Fig. 9. A Mcl-1 inhibitor attenuated ischemic kidney injury.

WT mice were pretreated with the Mcl-1 inhibitor, S63845 and sacrificed 16 h after the ischemic insult. A–D S63845 treatment led to decreases in numbers of kidney neutrophils but increases in neutrophil apoptosis evaluated with flow cytometry analysis of cleaved caspase 3 (n = 4) (A) and cleaved caspase-9 and S100A9 co-staining (4 and 5) (B), in association with less injury, indicated by lower BUN (n = 6 and 7) (C) and a lower tubular injury score (n = 7 and 8) (D). E Mcl-1 inhibition with S63845 led to increased ex vivo bone marrow neutrophil apoptosis determined by cleaved caspase-3 flow cytometric analysis (n = 4). Of note, cleaved caspase-3 neutrophil counts vs. total neutrophil counts are presented in parenthesis. Scale bar: 50 μm for (B) and 100 μm for (D). Data are means ± SEM, *P < 0.05, **P < 0.01, analyzed using 2 tailed Student’s t test for (A–D); 2-way ANOVA followed by Tukey’s post hoc test for (E).

Fig. 10. Mcl-1 inhibition accelerated functional recovery from ischemic injury with less post-ischemia fibrosis.

A Male C57BL/6 (8 weeks old) mice underwent ischemic injury (renal pedicle clamping, 35 min, both kidneys, Bi-IRI)), and the Mcl-1 inhibitor (S63845) was given 6 h later and daily for the next 5 days via intravenous injection at a dose of 5 mg/kg. The mice were sacrificed 3 weeks later. Mcl-1 inhibition accelerated functional recovery, indicated by: B rapid BUN decline (n = 9 and 10), C preserved GFR (n = 8) and attenuation of the development of kidney fibrosis, indicated by: D lower mRNA levels (n = 6 and 9) and E protein levels (n = 7) of profibrotic and fibrotic components as well as F quantitative Picrosirius red staining (n = 6) (F). Scale bar=100 μm. Data are means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, analyzed using 2 tailed Student’s t test for all.