Abstract
Defects in clearance of dying cells have been proposed to underlie the pathogenesis of systemic lupus erythematosus (SLE)1. Mice lacking molecules associated with dying cell clearance develop SLE-like disease2, and phagocytes from patients with SLE often display defective clearance and increased inflammatory cytokine production when exposed to dying cells in vitro. Previously, we3,4,5,6 and others7 described a form of noncanonical autophagy known as LC3-associated phagocytosis (LAP), in which phagosomes containing engulfed particles, including dying cells3,4,7, recruit elements of the autophagy pathway to facilitate maturation of phagosomes and digestion of their contents. Genome-wide association studies have identified polymorphisms in the Atg5 (ref. 8) and possibly Atg7 (ref. 9) genes, involved in both canonical autophagy and LAP3,4,5,6,7, as markers of a predisposition for SLE. Here we describe the consequences of defective LAP in vivo. Mice lacking any of several components of the LAP pathway show increased serum levels of inflammatory cytokines and autoantibodies, glomerular immune complex deposition, and evidence of kidney damage. When dying cells are injected into LAP-deficient mice, they are engulfed but not efficiently degraded and trigger acute elevation of pro-inflammatory cytokines but not anti-inflammatory interleukin (IL)-10. Repeated injection of dying cells into LAP-deficient, but not LAP-sufficient, mice accelerated the development of SLE-like disease, including increased serum levels of autoantibodies. By contrast, mice deficient in genes required for canonical autophagy but not LAP do not display defective dying cell clearance, inflammatory cytokine production, or SLE-like disease, and, like wild-type mice, produce IL-10 in response to dying cells. Therefore, defects in LAP, rather than canonical autophagy, can cause SLE-like phenomena, and may contribute to the pathogenesis of SLE.
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Change history
04 May 2016
An addition was made to the Acknowledgements.
28 September 2016
A Correction to this paper has been published: https://doi.org/10.1038/nature19837
31 August 2022
This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1038/s41586-022-05201-2
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Acknowledgements
The authors thank T. Brewer, P. Fitzgerald, M. Henderson, J. Kolb and T. Oguin for technical assistance. We also thank K. Gerrish and R. Fannin for their work and analysis of the Nanostring data. This work was supported by the Intramural Research Program of the National Institutes of Health, NIEHS (1ZIAES10328601), as well as grants from the US National Institutes of Health (RO1 AI40646, U19 AI109725), the Lupus Research Institute, the German Research Foundation (EXC306), and ALSAC.
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J.M. and D.R.G. designed the experiments; J.M. performed and analysed the experiments; L.D.C., S.P., M.Y., Q.L., Q.-Z.L., M.Y., L.J., C.G., A.L. and R.O. performed and analysed specific experiments; J.M., H.W.V. and D.R.G. wrote the manuscript.
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Extended data figures and tables
Extended Data Figure 1 LysM-Cre recombinase activity in vivo.
a, b, Bone marrow (a) and spleen (b) were obtained from wild-type and LysM-Cre+ LSL–YFP reporter mice (R26-stop-EYFP) at 8 weeks of age and flow cytometry was performed to examine expression of YFP in the following cellular populations: macrophages (CD11b+ F4/80+), neutrophils (CD11b+ Ly6G+), monocytes (CD11b+ CD115+), conventional dendritic cells (CD11b+ CD11c+), plasmacytoid dendritic cells (CD11cint B220+), CD8α+ dendritic cells (CD11c+ CD8α+), eosinophils (CD11b+ SiglecF+), B cells (CD19+ B220+), CD4+ T cells (CD3+ CD4+), and CD8+ T cells (CD3+ CD8+). Error bars represent s.d. (n = 4).
Extended Data Figure 2 Mice with LAP deficiencies display symptoms of immune activation.
a, Wild-type and deficient littermates were co-housed and aged for 52 weeks at SJCRH. Whole blood was collected at 52 weeks and analysed for differential blood count. Error bars represent s.d. LYM, lymphocytes; NEU, neutrophils; WBC, white blood cell s. b, c, Peripheral blood from Rubcn +/+ and Rubcn −/− animals aged 52 weeks was analysed for immune cell populations. Neutrophils (singlets/CD3− CD19−/Gr-1hi CD11b+), monocytes (singlets/CD3− CD19−/Gr-1int CD11b+), activated T cells (singlets/CD3+ CD4+/CD44+ CD62L− and singlets/CD3+ CD8+/CD44+ CD62L−), and central memory T cells (singlets/CD3+ CD4+/CD44+ CD62L+ and singlets/CD3+ CD8+/CD44+ CD62L+) were analysed and quantified. Error bars represent s.d. (n = 5, **P < 0.05, Student’s t-test). d, Spleens from wild-type and deficient littermates aged for 52 weeks were stained for anti-CD3 (top) or Ki67 (bottom) using immunohistochemistry. Representative images (original magnification, ×2.5) are shown (n = 4 per genotype). Error bars represent s.d. The colour scheme throughout represents LAP-deficient, autophagy-deficient genotypes (green), autophagy-deficient, LAP-sufficient (red), and autophagy-sufficient, LAP-deficient (blue).
Extended Data Figure 3 Mice with LAP deficiencies display increased levels of circulating autoantibodies.
Serum from animals aged 52 weeks at SJCRH was analysed for autoantigens commonly associated with autoimmune and autoinflammatory disorders. The background subtracted signal intensity of each autoantigen was normalized to the average intensity of the total mouse IgG, which was included on the array as an internal control. IgG autoantibodies are shown, in triplicates per genotype.
Extended Data Figure 4 Mice with LAP deficiencies display kidney pathology.
a, b, Wild-type and deficient littermates were co-housed and aged for 52 weeks at SJCRH. At 32 weeks, kidneys were obtained and stained for anti-IgG (red) and DAPI (blue) (a). Original magnification, ×100. MFI of anti-IgG staining in the glomeruli was calculated using Slidebook6 software (b). Error bars represent s.d. (n > 15 glomeruli per genotype, *P < 0.001, Student’s t-test). c, At 52 weeks, serum was collected and analysed for blood urea nitrogen (BUN). d, At 52 weeks, urine was collected, and proteinuria was calculated as the ratio of albumin to creatinine (ACR). Error bars represent s.d. (n > 4 per genotype, *P < 0.001, **P < 0.05). e, At 52 weeks, kidneys were obtained and stained for haematoxylin and eosin. Kidneys were scored blindly for endocapillary proliferative glomerulonephritis (EPG) on a scale of 1 (no damage) to 5 (clear damage). For histological assessment, at least 24 glomeruli were evaluated for each genotype. Error bars represent s.d. (*P < 0.001, Student’s t-test).
Extended Data Figure 5 Mice with LAP deficiencies display increased expression of the IFN signature but normal phagocytic capacity.
a, Wild-type and deficient littermates were co-housed and aged for 52 weeks at SJCRH. RNA was extracted from 52-week-old spleens and analysed for expression of genes associated with the IFN signature using Nanostring technology. Heatmap of Nanostring counts from the top 26 regulated genes in the IFN signature are shown in triplicate per genotype. b, UV-irradiated wild-type (WT) thymocytes were stained with CellTrace Violet and co-cultured (5:1) with bone-marrow-derived macrophages from wild-type and deficient genotypes for 45 min. Percentage phagocytosis (%CellTrace Violet+) was quantified by flow cytometry (singlets/GFP+ CellTrace Violet+). c, Wild-type and deficient littermates were co-housed and aged for 52 weeks at SJCRH. Peritoneal macrophages were isolated after 3 days of intra-peritoneal injection of thioglycolate. UV-irradiated wild-type thymocytes were stained with CellTrace Violet and co-cultured (2:1) with peritoneal macrophages from wild-type and deficient genotypes for 1 h. Phagocytic efficiency (singlets/CellTrace Violet+/F4/80+) was quantified by flow cytometry (%CellTrace Violet+). Error bars represent s.d. Data shown are representative of two independent experiments.
Extended Data Figure 6 Mice with LAP deficiencies display defective clearance of engulfed, dying cells.
a, 1 × 107 PKH26-labelled UV-irradiated wild-type thymocytes were injected intravenously into Cre− Atg7 f/f, Cre+ Atg7 f/f, Cre− Fip200 f/f, or Cre+ Fip200 f/f animals (all GFP–LC3+). Presence of labelled, apoptotic thymocytes was measured in kidney sections at 0, 24, 48, 72 and 96 h after transfer. Red cells are PKH26-labelled apoptotic thymocytes, and the kidney tissue is GFP–LC3. Representative images (original magnification, ×40) from two independent experiments are shown. b–d, Co-localization of lipidated GFP–LC3-II with engulfed dead cells was analysed by flow cytometry using digitonin treatment of spleen, liver and kidney of Cre− and Cre+ Atg7 f/f mice (b), Cre− and Cre+ Fip200 f/f mice (c), and Rubcn +/+ and Rubcn −/− mice (d) at the indicated time points. e, 1 × 107 PKH26-labelled UV-irradiated wild-type thymocytes were injected intravenously into wild-type, Rubcn −/−, or Tim4 −/− animals. After 24 and 48 h, spleens were collected and stained with fluorescently conjugated surface markers for macrophages (CD11b+ F4/80+), neutrophils (CD11b+ Gr-1+), monocytes (CD11b+ CD115+), and dendritic cells (CD11b+ CD11c+). Phagocytic efficiency of each cell type (singlets/cell surface markers+/PKH26+) was quantified by flow cytometry (percentage PKH26). Data shown are representative of two independent experiments. Error bars represent s.d. (**P < 0.05, *P < 0.001, Student’s t-test). The colour scheme represents LAP-deficient, autophagy-deficient genotypes (green), autophagy-deficient, LAP-sufficient (red), autophagy-sufficient, LAP-deficient (blue), and Tim4 +/+ and Tim4 −/− (black).
Extended Data Figure 7 LAP is required for the anti-inflammatory response to apoptotic cell engulfment in vitro.
a–d, UV-irradiated wild-type thymocytes were co-cultured with bone-marrow-derived macrophages from wild-type and deficient genotypes. Supernatant was collected at 24 h and analysed for IL-1β (a), IL-6 (b), IP-10 (c), and IL-10 (d) using Luminex technology. Error bars represent s.d. (n = 4, *P < 0.001, Student’s t-test). The colour scheme represents LAP-deficient, autophagy-deficient genotypes (green), autophagy-deficient, LAP-sufficient (red), autophagy-sufficient, and LAP-deficient (blue).
Extended Data Figure 8 Mice with LAP deficiencies display symptoms of an autoinflammatory disorder.
a–c, 2 × 107, UV-irradiated wild-type thymocytes were injected intravenously for 8 consecutive weeks into Rubcn +/+ or Rubcn −/− animals (aged 6 weeks). After 8 weeks, kidneys were obtained and stained with DAPI (blue), wheat germ agglutinin (green), anti-IgG (red, top) and anti-C1q (red, bottom) (a). Original magnification, ×100. MFI of anti-IgG (top) and anti-C1q (bottom) staining in the glomeruli was calculated using Slidebook6 software (b). Error bars represent s.d. (n > 15 glomeruli per genotype, *P < 0.001, Student’s t-test). After 8 weeks (week 8), serum was collected from uninjected and injected (+AT) animals (all 16 weeks of age) and analysed for alanine aminotransferase (ALT). Dots represent values from individual animals (c). Error bars represent s.e.m. (**P < 0.05, Student’s t-test). d–f, Wild-type and deficient littermates were co-housed and aged for 52 weeks at SJCRH. Serum was collected every 4 weeks and analysed for KC (d), MIP-1β (e), and MCP1 (f) using Luminex technology. Error bars represent s.d. The colour scheme throughout represents LAP-deficient, autophagy-deficient genotypes (green), autophagy-deficient, LAP-sufficient (red), and autophagy-sufficient, LAP-deficient (blue). Values for one cohort of Tim4 +/+ and Tim4 −/− animals are shown for comparison in all cases (black) in a–c.
Extended Data Figure 9 Mice with LAP deficiencies display symptoms of an autoinflammatory disorder.
Wild-type and deficient littermates were co-housed and aged for 52 weeks at Washington University. Serum was collected at 48–52 weeks and analysed for IL-1β (a), IL-6 (b), IL-12p40 (c), IP-10 (d), KC (e), MIP-1β (f), MCP1 (g), and IL-10 (h) using Luminex technology. Serum was analysed for anti-dsDNA antibodies (total Ig (i) and creatinine (j)). Error bars represent s.d. (**P < 0.001, Student’s t-test). The colour scheme throughout represents LAP-deficient, autophagy-deficient genotypes (green) and autophagy-deficient, LAP-sufficient (red).
Extended Data Figure 10 Mice with LAP deficiencies display increased levels of circulating autoantibodies.
Serum from animals aged 52 weeks at Washington University was analysed for autoantigens commonly associated with autoimmune and autoinflammatory disorders. IgG autoantibodies are shown, in duplicates per genotype.
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Martinez, J., Cunha, L., Park, S. et al. RETRACTED ARTICLE: Noncanonical autophagy inhibits the autoinflammatory, lupus-like response to dying cells. Nature 533, 115–119 (2016). https://doi.org/10.1038/nature17950
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DOI: https://doi.org/10.1038/nature17950
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