肾小管上皮细胞巨自噬/自噬激活保护急性肾损伤(AKI)。然而,免疫细胞自噬的作用,比如涉及巨噬细胞的,AKI尚不清楚。在这项研究中,我们发现巨噬细胞自噬是AKI期间的适应性反应,因为巨噬细胞特异性自噬缺陷(atg5-/-)小鼠表现出更高的血清肌酐,更严重的肾小管损伤,ADGRE1/F4/80+巨噬细胞浸润增加,在LPS或单侧缺血再灌注诱导的AKI期间,与WT小鼠相比,炎症因子的表达升高。atg5-/-巨噬细胞的过继转移进一步支持了这一点,但不是WT巨噬细胞,在氯膦酸盐脂质体诱导的巨噬细胞耗竭小鼠中引起更严重的AKI。在体外也获得了类似的结果,即缺乏Atg5的骨髓衍生巨噬细胞(BMDM)在响应LPS和IFNG时大大增加了促炎细胞因子的表达。机械上,我们发现atg5缺失显著上调TARM1(T细胞相互作用,髓样细胞上的激活受体1),而抑制TARM1抑制LPS-和IFNG诱导的atg5-/-RAW264.7巨噬细胞的炎症反应。E3泛素连接酶MARCHF1和MARCHF8泛素化TARM1并以自噬依赖性方式促进其降解,而MARCHF1和MARCHF8的功能结构域的沉默或突变消除了TARM1降解。此外,我们发现泛素化的TARM1从质膜内化到内体,然后通过泛素结合自噬受体TAX1BP1和SQSTM1募集到自噬-溶酶体途径进行降解。总之,巨噬细胞自噬通过MARCHF1-和MARCHF8介导的TARM1降解抑制肾脏炎症来保护AKI。
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.