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. 2022 Jul 7;13(7):587.
doi: 10.1038/s41419-022-05048-5.

sGRP78 enhances selective autophagy of monomeric TLR4 to regulate myeloid cell death

Zhenghao Wu #  1   2   3 , Zhuoshuo Xu #  1 , Xiaoqi Zhou  1 , Heli Li  1 , Liang Zhao  1 , Yibing Lv  1 , Yanyan Guo  1 , Guanxin Shen  1 , Yong He  4 , Ping Lei  5
Affiliations

sGRP78 enhances selective autophagy of monomeric TLR4 to regulate myeloid cell death

Zhenghao Wu et al. Cell Death Dis. .

Abstract

Soluble glucose regulated protein 78 (sGRP78) has long been suggested as a mediator resolution of inflammation. We previously reported that sGRP78 induced the rapid endocytosis of TLR4 with defective TLR4 signaling. To elucidate the underlying mechanisms, in this study, we investigated how sGRP78 influenced the behavior and trafficking of TLR4 in myeloid cells. It was found that sGRP78 promoted LPS endocytosis with monomeric TLR4. This internalized monomeric TLR4 formed complexes with p62-LC3, and was degraded in autolysosomes. Furthermore, the sGRP78-enhanced autophagy-dependent TLR4 degradation caused apoptosis and ferroptosis in myeloid cells, contributing to the sGRP78-mediated resolution of inflammation. These reports establish innovative mechanisms for endotoxin clearance and immune regulation by TLR4 degradation, linking innate immunity with multiple ancient processes, including autophagy, apoptosis, and ferroptosis, together through a shared resolution-associated molecular pattern (RAMP)-sGRP78.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. sGRP78 promotes endocytosis of LPS–TLR4 complex.
A RAW264.7 was treated with FITC-LPS at indicated concentrations for 1 h. B Cells were treated with GRP78 (20 μg/mL) and/or FITC-LPS (100 ng/mL) for 1 h. C, D To remove FITC-LPS fluorescence on the plasma membranes, cells in (B) were treated with acid-washing (C) or anti-FITC antibodies for fluorescence quenching (D). LPS binding was assessed by FCM. Representative histograms (left), and the MFI of FITC-LPS were quantified (right). E BMDCs and BMDMs F were treated with FITC-LPS for 0.5 h. FITC-LPS and stained TLR4 were detected by confocal microscopy (CLSM). Scale bar = 2 μm. The colocalization between FITC-LPS and TLR4 was described by 2D intensity histogram and Pearson's R value. G, H Immunoprecipitation assay for endogenous TLR4 with FITC-LPS or CD14. Data are representative of three independent experiments with similar results or mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2
Fig. 2. sGRP78 promotes endocytosis of monomeric TLR4 in a CD14-dependent manner.
BMDCs (A), BMDMs (B) and CD14 KO BMDCs (C) were treated with sGRP78 and/or LPS (100 ng/mL) for indicated minutes. Representative line chart of plasma membrane (upper) or intracellular (lower) TLR4 monomer were depicted. D Native PAGE for detection of the monomeric and dimeric TLR4. E Calculation of TLR4 monomer formation using the following formula: TLR4 monomer band/(TLR4 dimer band + TLR4 monomer band). F Tlr4−/− BMDCs were co-transfected with pHIS-TLR4 and pFLAG-TLR4 for 48 h. Then cells were treated with sGRP78 and/or LPS for 3 h. Cell extracts were immunoprecipitated with anti-FLAG, probed with anti-HIS. G Schematic description for the mechanism of detecting monomeric and dimerized TLR4 in TLR4-EGFP transfected Tlr4−/− BMDCs (left). LPS and GRP78-dependent polarization responses (right). Higher fluorescence polarization changes (△mP) indicate more monomer-dimer transition. Data are representative of 3 independent experiments with similar results or mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3
Fig. 3. sGRP78 promotes Atg7-dependent autophagy.
Cells were treated with sGRP78 (20 μg/ml) and/or LPS (0, 10, 100 ng/ml) for 12 h. Whole-cell lysates from BMDCs (A, C) or BMDMs (B, D) were subjected for immunoblot analysis of LC3, p62 (A, B) and Atg3, Atg7, Atg12, Atg16l1 (C, D). The upregulated autophagy proteins in GRP78-conditioned cells were highlighted in red in schematic diagram. E, F LC3, TLR4 and Atg7 expression in Wildtypeand Atg7-knockdown BMDCs. *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4. sGRP78 promotes selective autophagy of intracellular TLR4.
BMDCs (A) and BMDMs (B) were treated with sGRP78 and/or LPS for 12 h. Whole-cell lysates were immunoprecipitated with anti-LC3, anti-p62 or anti-TLR4 antibody, respectively. *P < 0.05, **P < 0.01.
Fig. 5
Fig. 5. sGRP78 promotes autophagosome formation in myeloid cells.
A–D 12 h after sGRP78 treatment, BMDCs (A, C) and BMDMs (B, D) were immunolabeled with LC3 (A, B) and p62 (C, D) antibody. BMDCs (E–H) or FITC-LPS-treated BMDCs (I) were incubated with sGRP78 for 1 h, followed by detection of intracellular LC3, p62, LPS, TLR4, and LAMP1. The colocalization between two markers was described by 2D intensity histogram and Pearson's R value. All images are representative of at least three independent experiments in which >100 cells were examined, and >95% of cells showed similar staining. Scale bar = 10 μm (A–D) or 2 μm (E–H). **P < 0.01, ****P < 0.0001.
Fig. 6
Fig. 6. sGRP78 promotes autophagy-dependent death of myeloid cells.
BMDCs (A) and BMDMs (B) were treated with LPS or Grp78 for 24 h followed by AnnexinV/PI staining, C PI staining (Scale bar = 10 μm) and D LDH release assay. E Cells were treated with LPS, Grp78, rapamycin (10 nM), or chloroquine (10 μM) for 24 h. Then cell death was assessed through PI staining by FCM. Data are mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 7
Fig. 7. sGRP78 promotes apoptosis and ferroptosis of myeloid cells.
A Cells were treated with LPS, Grp78, Casp3i (20 μM) and Fer-1 (2 μM) for 24 h. Then cell death was evaluated through PI staining by FCM. Apoptosis of sGRP78-conditioned cells was analyzed by Hoechst 33258 staining (B), DNA ladder (C) and Caspase 3 cleavage (D). E, F Cells were treated with Grp78, ROS positive control, Fer-1 and Erastin (10 μM). Intracellular and lipid ROS were detected. G Total iron concentration. H Transmission electron microscopy of BMDCs conditioned with sGRP78 for 48 h. White arrowheads, chromatin condensation; Black arrowheads, shrunken mitochondria; White arrow, the formation of double-membrane vesicles. A minimum of 10 cells per treatment condition was examined. Data are representative of three independent experiments with similar results or mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Fig. 8
Fig. 8. sGRP78-promoted myeloid cells death is TLR4 dependent.
BMDCs and BMDMs from WT and TLR4 KO mouse were treated with Grp78 for 24 h. Apoptosis were assayed by AnnexinV/7-AAD staining (A, B) and by Hoechst 33258 staining (C). Scale bar = 10 μm. D–G Intracellular ROS (D, E) and lipid ROS (F, G). Data are mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. H Schematic description for the role of sGRP78 (HSPA5) in inducing selective autophagy of monomeric TLR4 to regulate myeloid cell death.
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