Abstract
The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.
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Acknowledgements
We thank F. Barrat (Weill Cornell College of Medicine) for the TLR inhibitor and control oligonucleotides; M. Shlomchik, A. Marinov and Z. Rahman for assistance with the analysis of SLE-prone mice; N. Winegarden and the Princess Margaret Genomics Centre for assistance with sequence analysis; K. Hultenby and B. Calvieri for performing electron microscopy; J.C. Zuniga-Pflucker for advice during development of the research report; M. Butler for assistance with the acquisition of PBMCs from healthy control subjects; and P. Ohashi and D. Brooks for reading and critiquing the manuscript. Supported by the US National Institutes of Health (AI105500, AR067763 and CA190449), the Medicine by Design/Canada First Research Excellence Fund (T.L.M.), the Swedish Medical Research Council and the Karolinska Institute (S.G.).
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R.S., K.H., R.D., M.E., S.L., D.W. and S.G. executed the biochemical, cell biological and in vitro experiments; R.S., M.J.H., B.R., H.L. and K.C. performed the animal experiments; A.K. and A.T. analyzed the RNA-seq results; R.S., A.C., T.d.S.M. and D.D.D.C. performed the ATAC-seq experiments and analysis; M.M. assigned scores for renal pathology; K.P.M., Y.B., M.M., S.B.C., D.H.M., S.G., Z.T. and J.W., contributed reagents and human samples and to discussions; R.S., K.H., M.J.H., A.K., A.C. and T.L.M. prepared figures; R.S. and T.L.M. wrote the paper; and T.L.M. designed and supervised the research.
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Supplementary Figure 1 Apoptotic cell activation of AhR is contact and phagocytosis dependent.
(a) BMDC of the indicated genotype were co-cultured with B6 apoptotic thymocytes (Ap-BMDC) for 8h and indicated mRNA species were measured by sqPCR. (b) BMDM were cultured with apoptotic cells in a trans-well or in direct contact (Ap-BMDM) and measured at the indicated time points for Cyp1a1 expression by sqPCR. (c) Ap-BMDM were cultured with 1μM cycloheximide (CHX) and Cyp1a1 mRNA was measured by sqPCR. (d) BMDM were cultured with apoptotic or necrotic cells (3x freeze/thawed) as in b and Cyp1a1 mRNA was quantified by sqPCR. (e) BMDM were cultured with live or apoptotic thymocytes and Cyp1a1 mRNA was measured after 6h by sqPCR. Times indicate culture period post-irradiation prior to addition to BMDM culture. (f) Ap-BMDM were co-cultured with CFSE-labeled apoptotic cells for 2h and assessed efferocytosis by flow cytometry. Cytochalsin B was used in the co-culture assay to block phagocytosis. To mask phosphatidylserine, apoptotic cells were incubated with purified Annexin V at 50 μg/ml for 10 min prior to co-culture. Pan-caspase inhibitor z-vad(ome)-fmk was used to inhibit apoptosis in thymocytes prior to addition to BMDM cultures. (g) Ap-BMDM described in f were measured for induction of Cyp1a1 mRNA by sqPCR. For a to e,g values were normalized against β-actin. *=P≤ 0.05, **=P≤ 0.01 as determined by two-sided Student’s t-test. For all experiments n=3 biologically independent samples per group. Bars are mean value +/- standard deviation. Experiments were repeated three times with similar results.
Supplementary Figure 2 Diseases and functional pathways predicted by IPA to be differentially regulated after inhibition of AhR signaling.
IPA analysis demonstrating significantly enriched diseases and functions separated by categories and functions. Bars represent activation z-scores of given process for Ap-BMDM in the presence (shown on left) or absence (shown on right) of the AhR inhibitor CH223191.
Supplementary Figure 3 Apoptotic cell-driven AhR induction is independent of IDO and driven by TLR9.
(a) mRNA for Ido1 was measured by sqPCR in BMDM and Ap-BMDM. (b) Culture supernatants from BMDM cultures treated as described in a were assessed for IDO enzymatic activity (i.e. increased tryptophan conversion to kynurenine) by HPLC. (c) Cyp1a1 message was measured in BMDM and Ap-BMDM cultures by sqPCR. Groups of Ap-BMDM included B6.Ido1−/− or B6 +/- the IDO-inhibitor D-1-methyl-tryptophan (200μm). (d) Ap-BMDM cultures were done in the presence of oligonucleotide inhibitors of TLR7 (IRS 661), TLR9 (IRS 869), or TLR7/9 (IRS 954) (1μM for all inhibitors). Cyp1a1 expression was measured by sqPCR. For a,b,d,d n=3 biologically independent samples per group. Bars and data points in line graphs are mean values for group +/- standard deviation. *=P≤ 0.05, ns= not significant as determined by two-sided Student’s t-test. For sqPCR samples were normalized against Bactin. (e) Full image for immuno-blots shown in Fig. 1c. (f) Full image for immuno-blots shown in Fig. 3b. (g) Full image for immuno-blots shown in Fig. 3d. All Experiments were repeated three times with similar results.
Supplementary Figure 4 FACS sorting strategy for purification of splenic myeloid cell populations from mice and PBMC myeloid cell populations from SLE patients and healthy controls.
(a) Mouse splenic macrophages and dendritic cells were sorted on the basis of the markers indicated. For DC sorting we included the marker CD103 which is associated with DC that have significant efferocytosis activity. DC-dendritic cell, pDC- plasmacytoid DC. (b) Strategy for sorting of human myeloid cells from PBMC fractions. Lineage markers used to exclude non-myeloid cells were CD56, CD3, and CD19. cDC- conventional myeloid DC, pDC- plasmacytoid DC. In a separate sort CD3, CD4, and CD8 were used to enrich for CD4+ and CD8+ T cells.
Supplementary Figure 5 AhR does not impact dexamethasone-induced thymic involution and Cyp1a1 expression is independent of Ido1 and the microbiome in myeloid cells from lupus prone mice.
(a) Thymic cell numbers were quantified in B6 mice, B6 mice treated with AhR inhibitor (CH223191), and LysM-AhR cKO mice 24h after dexamethasone administration (0.2 mg per mouse, i/p). Bar graph represents total thymic cell count. (b, c) DC and macrophages were FACS-sorted from the spleen of female, 12 week old mice of the mouse strain indicated on the basis of F4/80, CD11c, CD8α. Expression of mRNA for Cyp1a1 and Ido1 were measured by sqPCR. For a, b, c n=4 biologically independent samples per group. Bars represent the mean +/- the standard deviation. (d) 12 week old female R2B mice were kept on antibiotic water for two weeks. DC and macrophages were sorted as in b and mRNA for Cyp1a1 and Ido1 were measured. Bars represent the value of pooled samples from four mice. For all sqPCR analysis samples were normalized against Bactin. ***=P<0.001 as determined by two-sided Student’s t-test. All experiments were repeated two times with similar results.
Supplementary Figure 6 AhR limits autoimmunity in lupus prone mice.
(a,b) 8w-old R2B mice were treated with ITE and CH223191. After 8w of treatment splenocytes were assessed by cytometry analysis. (a) Cytometry plots gated on B220+ B cells measuring CD24. (b) Flow cytometry analysis of CD4+CD44high and CD8+CD44high T cell percentages. (c) 8w female R2B mice treated with ITE (AhR agonist), CH223191 (AhR antagonist), or vehicle for 4m. B6 mice were age/sex matched and treated with vehicle. IgG antinuclear antibody (ANA) reactivity was measured. Scale bar=50μm. (d) Immunoglobulin auto-reactivity against dsDNA and histones from the mice in c was measured by ELISA. (e) Kidneys were collected from mice in c, 5μm sections were stained with H&E and pathology of glomeruli and tubules was scored in a blinded manner. Scale bar=100μm. For all experiments n=5 biologically independent samples per group. For all graphs bars are mean values +/- standard deviation. *=P≤ 0.05, **=P≤ 0.01 and ns= not significant as determined by two-sided Student’s t-test. Experiments were repeated three times with similar results.
Supplementary Figure 7 Apoptotic cells and SLE microparticles drive AhR activation and IL-10 production in human macrophages.
(a) Irradiated, apoptotic jurkat cells were cultured with PBDM (Ap-PBDM) for 8h. Culture supernatants were collected and IL-10 and IL-6 were measured by ELISA and mRNA was collected for assessment of CYP1A1 (normalized against BACTIN) by sqPCR. (b) Staurosporine treated, apoptotic jurkat cells were treated with DNAse and cultured with PBDM for 8h (Ap-PBDM). Culture supernatants were collected indicated proteins were measured by ELISA and mRNA was collected for assessment of CYP1A1 sqPCR. (c) Serum concentrations of kynurenine (Kyn) and indole-3-propionic acid (IPA) were measured by HPLC from samples described in Supplementary Tables 1,2,3. Bars are mean +/- standard deviation. *=P≤ 0.05, ns= not significant as determined by the Wilcoxon rank-sum test. (d) Microparticles purified from plasma of patient samples described in Supplementary Table 4,5 were quantified by flow cytometry. (e) Representative electron micrographs illustrating morphology and size distribution of microparticles from plasma of SLE patients and controls. Bar is 500nm and magnification is 50,000x. (f) Microparticles were assessed by flow cytometry for surface expression of the markers indicated. MFI ratio represents the relative mean fluorescence intensity (MFI) for the indicated CD marker versus the MFI for isotype controls. (g) Microparticls origin was analyzed by expression of endothelial (CD31), haematopoietic (CD45), neutrophil (CD66b), B cell (CD19), and T cell (CD3) markers. % positive indicates the % of microparticles that were positive for the markers compared to isotype controls. (h) Microparticles isolated from the plasma of SLE patients were added to cultures of healthy donor PBDM (20% volume/volume) +/- AhR antagonist CH223191. Eight hours later, culture supernatants were collected to measure cytokine production by ELISA. For a, b, h n=5 biologically independent samples per group. Bars are mean values +/- standard deviation and *=P≤0.05, **=P≤0.01 as determined by two sided Student’s t-test. All Experiments were repeated three times with similar results.
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Shinde, R., Hezaveh, K., Halaby, M.J. et al. Apoptotic cell–induced AhR activity is required for immunological tolerance and suppression of systemic lupus erythematosus in mice and humans. Nat Immunol 19, 571–582 (2018). https://doi.org/10.1038/s41590-018-0107-1
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DOI: https://doi.org/10.1038/s41590-018-0107-1
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