Macrophage autophagy protects against acute kidney injury by inhibiting renal inflammation through the degradation of TARM1

Abstract

Macroautophagy/autophagy activation in renal tubular epithelial cells protects against acute kidney injury (AKI). However, the role of immune cell autophagy, such as that involving macrophages, in AKI remains unclear. In this study, we discovered that macrophage autophagy was an adaptive response during AKI as mice with macrophage-specific autophagy deficiency (atg5^-/-) exhibited higher serum creatinine, more severe renal tubule injury, increased infiltration of ADGRE1/F4/80⁺ macrophages, and elevated expression of inflammatory factors compared to WT mice during AKI induced by either LPS or unilateral ischemia-reperfusion. This was further supported by adoptive transfer of atg5^-/- macrophages, but not WT macrophages, to cause more severe AKI in clodronate liposomes-induced macrophage depletion mice. Similar results were also obtained in vitro that bone marrow-derived macrophages (BMDMs) lacking Atg5 largely increased pro-inflammatory cytokine expression in response to LPS and IFNG. Mechanistically, we uncovered that atg5 deletion significantly upregulated the protein expression of TARM1 (T cell-interacting, activating receptor on myeloid cells 1), whereas inhibition of TARM1 suppressed LPS- and IFNG-induced inflammatory responses in atg5^-/- RAW 264.7 macrophages. The E3 ubiquitin ligases MARCHF1 and MARCHF8 ubiquitinated TARM1 and promoted its degradation in an autophagy-dependent manner, whereas silencing or mutation of the functional domains of MARCHF1 and MARCHF8 abolished TARM1 degradation. Furthermore, we found that ubiquitinated TARM1 was internalized from plasma membrane into endosomes, and then recruited by the ubiquitin-binding autophagy receptors TAX1BP1 and SQSTM1 into the autophagy-lysosome pathway for degradation. In conclusion, macrophage autophagy protects against AKI by inhibiting renal inflammation through the MARCHF1- and MARCHF8-mediated degradation of TARM1.Abbreviations: AKI, acute kidney injury; ATG, autophagy related; Baf, bafilomycin A₁; BMDMs, bone marrow-derived macrophages; CCL2/MCP-1, C-C motif chemokine ligand 2; CHX, cycloheximide; CQ, chloroquine; IFNG, interferon gamma; IL, interleukin; IR, ischemia-reperfusion; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; MARCHF, membrane associated ring-CH-type finger; NC, negative control; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; NLRP3, NLR family, pyrin domain containing 3; NOS2, nitric oxide synthase 2, inducible; Rap, rapamycin; Wort, wortmannin; RT-qPCR, real-time quantitative polymerase chain reaction; Scr, serum creatinine; SEM, standard error of mean; siRNA, small interfering RNA; SYK, spleen tyrosine kinase; TARM1, T cell-interacting, activating receptor on myeloid cells 1; TAX1BP1, Tax1 (human T cell leukemia virus type I) binding protein 1; TECs, tubule epithelial cells; TNF, tumor necrosis factor; WT, wild type.

Keywords: Adoptive transfer; Atg5; bone marrow-derived macrophages; inflammation; ischemia-reperfusion; tubular epithelial cells.

Figure 1.

Autophagy is an adaptive response in renal macrophages in AKI. (A) expression of autophagic genes of single-cell transcriptome from the kidneys of mice subjected to IR for 3 days. (B) immunofluorescence staining of CD68 and LC3 in the kidney biopsy of patients with AKI. (C and D) immunofluorescence staining of ADGRE1 and tandem-tagged mCherry-EGFP-LC3 in the kidneys of mice subjected to IR for 3 days or LPS (5 mg/kg) for 24 h (white arrowheads indicating mCherry-EGFP-LC3, orange arrowheads indicating mCherry-LC3). Scale bars: 15 μm or 5 μm.

Figure 2.

Mice with macrophage autophagy deficiency develop more severe AKI induced by LPS or IR. (A) the survival curves of female WT mice and female atg5^−/−mice treated with LPS (5 mg/kg) treatment (n = 10). (B) serum levels of creatinine in WT mice and atg5^−/− mice treated with or without LPS (1 mg/kg) for 24 h. (C and D) representative histology in the renal cortex by H&E staining and immunohistochemical staining of HAVCR1 in renal tissues of mice with SA-AKI. (n = 7-10 per group). (E and F) representative western blot images and quantification of HAVCR1 in renal tissues of mice with SA-AKI. (n = 4-6 per group). (G and H) pathological observation of kidney tissue by H&E staining and immunohistochemical staining of HAVCR1 in renal tissues of mice subjected to IR for 3 days. (n = 5-7 per group) (I and J) Representative western blot images and quantification of HAVCR1 in renal tissues of mice subjected to IR for 3 days. (n = 4-7 per group) all data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test. Scale bars: 50 μm.

Figure 3.

Macrophage autophagy deficiency enhances renal inflammation in mice with AKI. (A and B) immunofluorescence and quantification of ADGRE1⁺ macrophages in renal tissues of mice with SA-AKI. (n = 6-10 per group). (C and D) Representative western blot images and quantification of pro-inflammatory CCL2, NLRP3, and IL18 in the renal tissues of mice treated with LPS (1 mg/kg) for 24 h. (n = 4-6 per group). (E and F) immunofluorescence and quantification of ADGRE1⁺ macrophages in renal tissues of mice subjected to IR for 3 days. (n = 5-7 per group). (G and H) Representative western blot images and quantification of pro-inflammatory CCL2, NLRP3, and IL18 in the renal tissues of mice with ir-induced AKI. (n = 4-7 per group). (I and J) Representative western blot images and quantification of pro-inflammatory cytokines, NOS2, NLRP3, and IL18, in BMDMs subjected to LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. (n = 3 per group). All data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (B, D, F, H); unpaired Student’s t-test (J). Scale bars: 50 μm.

Figure 4.

Adoptive transfer of autophagy-deficient macrophages promotes SA-AKI in clodronate liposomes-mediated macrophage depletion mice. (A) schematic diagram of administration of clodronate liposomes to deplete macrophages and adoptive transfer macrophages in mice. (B) quantification of serum creatinine levels in SA-AKI-bearing mice that received atg5^−/− or WT RAW 264.7 macrophage transfer. (C-D) Representative images of H&E, immunohistochemical staining, and quantification of HAVCR1 in renal tissues of SA-AKI-bearing mice that received atg5^−/− or WT RAW 264.7 macrophage transfer. (n = 5 per group). (E-I) Representative images of H&E, immunohistochemical staining of HAVCR1 in renal tissues, and serum levels of creatinine of SA-AKI-bearing mice that received atg5^−/− or WT BMDMs transfer. (n = 3 per group). (J) Representative western blot images of HAVCR1, IL18, NLRP3, and CCL2 expression in renal tissues of SA-AKI-bearing mice that received atg5^−/− or WT RAW 264.7 macrophage transfer. (n = 5 per group). (K) Representative western blot images of HAVCR1, IL18, NLRP3, and CCL2 expression in renal tissues of SA-AKI-bearing mice that received atg5^−/− or WT BMDMs transfer. (n = 3 per group). All data are presented as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test (B, C); and unpaired Student’s t-test (G-J). Scale bars: 50 μm.

Figure 5.

Macrophage autophagy suppresses inflammatory cytokine production by targeting TARM1. (A) proteomic analysis of the differential protein expression in WT and atg5^−/− BMDMs in response to LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. (n = 3 per group). (B and C) Representative western blot images and quantification of TARM1 expression in WT and atg5^−/− BMDMs in response to LPS and IFNG for 24 h. (n = 3 per group). (D and E) Representative western blot images and quantification of TARM1 expression in renal tissues of mice with lps-induced AKI. (n = 4-6 per group). (F and G) Representative western blot images and quantification of pro-inflammatory cytokines in WT and atg5^−/− RAW 264.7 macrophages transfected with siTarm1 or NC for 24 h then in response to LPS and IFNG for 24 h. (n = 4 per group). (H and I) flow cytometry and quantification of NOS2 level in WT and atg5^−/− RAW 264.7 macrophages transfected with siTarm1 or NC for 24 h then in response to LPS and IFNG for 24 h. (n = 4 per group). (J and K) flow cytometry and quantification of NOS2 level in WT and atg5^−/− RAW 264.7 macrophages pretreated with TARM1-FC (10 μg/mL) or IgG-fc (10 μg/mL) for 1 h then in response to LPS and IFNG for 24 h. (n = 4 per group). (L and M) Representative western blot images and quantification of pro-inflammatory cytokines in WT and atg5^−/− RAW 264.7 macrophages pretreated with Piceatannol (20 μM) or JSH-23 (20 μM) for 1 h then in response to LPS and IFNG for 24 h. (n = 4 per group). All data are presented as mean ± SEM. Unpaired Student’s t-test (C); two-way ANOVA followed by Tukey’s post hoc test (E, G, M); one-way ANOVA followed by Tukey’s post hoc test (I, K).

Figure 6.

Autophagy regulates TARM1 degradation in macrophages. (A and B) Representative western blot images and quantification of TARM1 in WT and atg5^−/− RAW 264.7 macrophages pretreated with LPS (100 ng/mL) and IFNG (50 ng/mL) for 12 h then stimulated with CHX (500 μg/mL) for 0 h, 2 h, 4 h, and 8 h. (n = 3 per group). (C and D) Representative western blot images and quantification of TARM1 in RAW 264.7 macrophages pretreated with MG132 (1 μM) for 1 h then in response to LPS and IFNG for 24 h. (n = 4 per group). (E and F) Representative western blot images and quantification of TARM1 in BMDMs pretreated with rap (10 μM), torin1 (1 μM), CQ (10 μM), and Baf (25 nM) for 1 h then in response to LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. (n = 4 per group). (G and H) Representative western blot images and quantification of TARM1 in WT and atg5^−/− RAW 264.7 macrophages pretreated with CQ (10 μM) for 1 h then in response to LPS and IFNG for 24 h. (n = 4 per group). (I and J) Representative western blot images and quantification of TARM1 in RAW 264.7 macrophages pretreated with wort (10 μM), LY294002 (10 μM), and CQ (10 μM) for 1 h then in response to LPS and IFNG for 24 h. (n = 4 per group). All data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (B, H); unpaired Student’s t-test (D); one-way ANOVA followed by Tukey’s post hoc test (F, J).

Figure 7.

E3 ubiquitin ligases MARCHF1 and MARCHF8 mediate TARM1 degradation. (A) schematic diagram of our hypothesized process of TARM1 degradation through autophagy. (B) prediction of E3 ubiquitin ligase that mediates TARM1 degradation. (C) Representative western blot images demonstrate a dose-dependent degradation of TARM1 by MARCHF1, MARCHF8, and SYTL4 in HEK293T cells co-transfected with TARM1-flag and MARCHF1-MYC, MARCHF8-MYC, or SYTL4-MYC plasmid for 24 h. (D) Representative western blot images of TARM1 in HEK293T cells were co-transfected with TARM1-flag and either MARCHF1-MYC or MARCHR8-MYC plasmids for 18 h, followed by treatment with CQ (40 μM) or MG132 (10 μM) for 6 h. (E) Representative western blot images demonstrate the interaction between TARM1 and either MARCHF1 or MARCHF8 proteins in HEK293T cells co-transfected with TARM1-flag, MARCHF1-MYC, and MARCHF8-MYC plasmids for 24 h. Cellular lysates were subjected to immunoprecipitation (IP) using anti-flag, followed by western blot with anti-myc and anti-flag. (F) Representative western blot images demonstrate the ubiquitination of TARM1 in HEK293T cells co-transfected with TARM1-his, HA-Ub, and either MARCHF1-MYC or MARCHF8-MYC plasmids for 18 h, followed by treatment with CQ for 6 h. Cellular lysates were purified with a his-tag protein purification kit and then western blot with anti-ha, anti-his, and anti-myc. (G) Representative western blot images demonstrate the ubiquitination of TARM1 in HEK293T cells co-transfected with TARM1-his, HA-Ub, and either WT MARCHF1-MYC (WT), MARCHF1[Mut]-myc (mut), WT MARCHF8-MYC (WT), and MARCHF8^Mut-MYC (mut) plasmids for 24 h. Cellular lysates were purified with a his-tag protein purification kit and then western blot with anti-ha, anti-his anti-myc.

Figure 8.

E3 ubiquitin ligases MARCHF1 and MARCHF8 inhibit the expression of TARM1 and attenuate the inflammatory response in macrophages. (A and B) Representative western blot images of MARCHF1 and MARCHF8 in RAW 264.7 macrophages silencing MARCHF1 and MARCHF8 by shMarchf1 and shMarchf8 lentivirus. (C and D) Representative western blot images and quantification of TARM1, NOS2, and IL18 in RAW 264.7 macrophages silencing MARCHF1 and MARCHF8 in response to LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. (n = 3 per group). (E and F) Representative western blot images of MARCHF1 and MARCHF8 in RAW 264.7 macrophages overexpressing MARCHF1-MYC and MARCHF8-myc. (G and H) Representative western blot images and quantification of TARM1, NOS2, and IL18 in RAW 264.7 macrophages overexpressing MARCHF1-MYC and MARCHF8-myc in response to LPS and IFNG for 24 h. (n = 3 per group). All data are presented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc test.

Figure 9.

Ubiquitylation is required for endocytosis of TARM1. (A) Representative western blot images demonstrate the ubiquitination of TARM1 in HEK293T cells co-transfected with WT TARM1-his (WT), TARM1[Mut]-his (mut), HA-Ub, MARCHF1-MYC, and MARCHF8-MYC plasmids for 24 h. Cellular lysates were purified with a his-tag protein purification kit and then western blot with anti-ha, anti-his anti-myc. (B) Representative images of immunofluorescence staining of his and EEA1 in RAW 264.7 macrophages overexpressing TARM1-his in response to LPS and IFNG for 24 h (arrowheads indicating TARM1 and EEA1 colocalization). Scale bars: 15 μm or 5 μm. (C and D) Representative western blot images and quantification of NOS2, NLRP3, and IL18 in RAW 264.7 macrophages overexpressing WT TARM1-his or TARM1[Mut]-his in response to LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. (n = 3 per group). Two-way ANOVA followed by Tukey’s post hoc test.

Figure 10.

Autophagy receptors TAX1BP1 and SQSTM1 are recruited to ubiquitylated TARM1 and mediate autophagosome formation. (A) Representative western blot images demonstrate the interaction between TARM1 and either NBR1, TAX1BP1, OPTN, SQSTM1 or CALCOCO2/NDP52 proteins in RAW 264.7 macrophages overexpressing TARM1-flag, followed by treatment with LPS (100 ng/mL) and IFNG (50 ng/mL) for 24 h. Cellular lysates were subjected to immunoprecipitation using anti-flag, followed by western blot with anti-NBR1, anti-TAX1BP1, anti-optn, anti-SQSTM1, anti-CALCOCO2/NDP52, and anti-flag. (B-E) Representative images and quantification of immunofluorescence staining of flag, TAX1BP1, SQSTM1, and NBR1 in RAW 264.7 macrophages overexpressing TARM1-flag in response to LPS and IFNG for 24 h (arrowheads indicating TARM1 and TAX1BP1, SQSTM1, or NBR1 colocalization). (F-J) Representative images and quantification of immunofluorescence staining of TARM1-flag and LC3 in RAW 264.7 macrophages simultaneously overexpressing both TARM1-flag and silencing TAX1BP1, SQSTM1, or NBR1 in response to LPS and IFNG for 24 h (arrowheads indicating TARM1 and LC3 colocalization). (K) Representative western blot images demonstrate the binding of ubiquitinated TARM1 to TAX1BP1 and SQSTM1 in HEK293T cells co-transfected with TARM1-HA, his-ub, TAX1BP1-strep-flag, SQSTM1-strep-flag, MARCHF8-MYC and empty vector plasmids for 18 h, followed by treatment with CQ for 6 h. The colocalization was monitored using Pearson’s correlation coefficient of the ImageJ software. Scale bars: 15 μm or 5 μm.

Figure 11.

The proposed schematic diagram for macrophage TARM1 degradation during AKI. Upon LPS or other risk factors stimulation, TARM1 is upregulated and stimulates proinflammatory cytokine production by renal macrophages to induce AKI. Meanwhile, E3 ubiquitin ligases MARCHF1 and MARCHF8 can ubiquitylate and degrade TARM1 via the autophagy receptors TAX1BP1 and SQSTM1-dependent mechanism, which attenuates macrophage-mediated renal inflammation in AKI. In contrast, autophagy deficiency in macrophages promotes macrophage-mediated AKI.