Abstract
At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA and self-DNA, respectively. Despite the structural and functional similarities between these receptors, their contributions to autoimmune diseases such as systemic lupus erythematosus can differ. For example, TLR7 and TLR9 have opposing effects in mouse models of systemic lupus erythematosus—disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice1. However, the mechanisms of negative regulation that differentiate between TLR7 and TLR9 are unknown. Here we report a function for the TLR trafficking chaperone UNC93B1 that specifically limits signalling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. Mutations in UNC93B1 that lead to enhanced TLR7 signalling also disrupt binding of UNC93B1 to syntenin-1, which has been implicated in the biogenesis of exosomes2. Both UNC93B1 and TLR7 can be detected in exosomes, suggesting that recruitment of syntenin-1 by UNC93B1 facilitates the sorting of TLR7 into intralumenal vesicles of multivesicular bodies, which terminates signalling. Binding of syntenin-1 requires phosphorylation of UNC93B1 and provides a mechanism for dynamic regulation of TLR7 activation and signalling. Thus, UNC93B1 not only enables the proper trafficking of nucleic acid-sensing TLRs, but also sets the activation threshold of potentially self-reactive TLR7.
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Data availability
The data that support the findings of this study are either included within the manuscript or are available from the corresponding author on reasonable request. Source Data for Figs. 1–4 and Extended Data Figs. 1–9 are included in the online version of the paper. Gel source data can be found in Supplementary Fig. 1.
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Acknowledgements
We thank R. Vance and members of the Barton and Vance laboratories for discussions and critical reading of the manuscript, A.Yiu-fai Lee and the Gene Targeting Facilty of the Cancer Research Center at UC Berkeley for generating the Unc93b1PKP/PKP knock-in mice, L. Kohlstaedt and the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at UC Berkeley for identification of UNC93B1-interacting proteins, H. Nolla and A. Valeros for assistance with cell sorting at the Flow Cytometry Facility of the Cancer Research Laboratory at UC Berkeley, and S. Ruzin and D. Schichnes for assistance with microscopy on the Zeiss Elyra PS.1 at the Biological Imaging Center at UC Berkeley. This work was supported by the National Institutes of Health (NIH; AI072429, AI105184 and AI063302 to G.M.B.) and by the Lupus Research Institute (Distinguished Innovator Award to G.M.B.). O.M. was supported by an Erwin Schrödinger (J 3415-B22) and CRI Irvington postdoctoral fellowship. B.L. was supported by the UC Berkeley Tang Distinguished Scholars Program. Research reported in this publication was supported in part by the NIH S10 program under award number 1S10OD018136-01 and by the NIH S10 Instrumentation Grant S10RR025622.
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O.M., B.L. and G.M.B designed experiments. O.M. and B.L. performed experiments and analysed the data for all figures. L.S.M.K. performed B cell stimulations. N.K. assisted with design of mass spectrometry experiments. G.M.B. wrote the manuscript. O.M, B.L. and G.M.B. revised and edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 A C-terminal region in UNC93B1 regulates TLR7 responses.
a, UNC93B1PKP-expressing macrophages show enhanced TLR7 signalling. Immunoblot of phosphorylated (P)-p38, P-JNK, P-ERK and IκBα of RAW macrophages stimulated with R848 (50 ng ml−1) for the indicated times. Data are representative of two independent experiments. b, UNC93B1–Flag expression levels, as measured by Flag immunoblot, of UNC93B1-deficient RAW macrophages retrovirally transduced to express the indicated UNC93B1 alleles. Cell lines are as described in Fig. 1a. All data are representative of three independent experiments, unless otherwise noted.
Extended Data Fig. 2 UNC93B1PKP does not alter TLR9 responses, unlike UNC93B1(D34A).
a, Representative flow cytometry analysis showing the percentage of TNF-positive cells, measured by intracellular cytokine staining, of indicated RAW macrophage lines after stimulation with CpG-B (25 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (20 µg ml−1), or LPS (10 ng ml−1). Shaded histograms show unstimulated controls. b, TNF production, measured by ELISA, from the indicated RAW macrophage lines after stimulation for 8 h with R848 (10 ng ml−1), CpG-B (25 nM) or LPS (50 ng ml−1). Data are mean of n = 4 biological replicates pooled from two independent experiments. P values determined by unpaired two-tailed Student’s t-test. c, TLR7 and TLR9 trafficking are normal in UNC93B1PKP but not in UNC93B1(D34A) RAW lines. Immunoblot of TLR7–HA and TLR9–HA from lysates of indicated RAW macrophage lines. FL, full-length. All data are representative of three independent experiments.
Extended Data Fig. 3 UNC93B1PKP does not alter TLR7 trafficking or localization.
a, UNC93B1PKP does not alter TLR7 export rates. Pulse-chase analysis of TLR7 in RAW macrophages expressing wild-type UNC93B1 and UNC93B1PKP. Cell lysate was immunoprecipitated with haemagglutinin and subjected to a radiolabelled screen and immunoblot. The full-length and cleaved forms of TLR7 are indicated. Asterisk denotes nonspecific band. Data are representative of two independent experiments. b, UNC93B1PKP does not affect TLR7 trafficking to endosomes. Levels of TLR7, LAMP1 and calnexin in whole-cell lysates or lysates of purified phagosomes from the indicated RAW macrophage lines were measured by immunoblot. Representative of three independent experiments. c, Colocalization of TLR7–HA (red) and LAMP1 (green) in RAW macrophages expressing the indicated UNC93B1–Flag alleles on a Myd88−/− background using super-resolution structured illumination microscopy. Boxed areas are magnified. The plot shows quantification of the percentage of total TLR7 within LAMP1+ endosomes, with each dot representing an individual cell. Data are mean ± s.d. and pooled from two independent experiments. Scale bars, 10 µm. P values determined by unpaired two-tailed Student’s t-test. d, The subcellular localization of UNC93B1PKP is not altered relative to wild-type UNC93B1. Co-localization of UNC93B1–Flag (red) and LAMP1 (green) was measured using super-resolution structured illumination microscopy in UNC93B1-deficient RAW macrophages complemented with wild-type, PKP or H412R mutant UNC93B1. A representative cell is shown for each UNC93B1 allele. Boxed areas are magnified. The plot shows quantification of the percentage of total UNC93B1 within LAMP1+ endosomes, with each dot representing an individual cell. Data are mean ± s.d. and acquired in a single experiment. Scale bars, 10 µm. P values determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 4 Mass spectrometry analysis of UNC93B1 complexes.
a, A small fraction of UNC93B1 resides in endosomes compared to the ER. Subcellular fractionation of RAW macrophages expressing TLR7–HA and UNC93B1–Flag was performed by density-gradient centrifugation. The distribution of calnexin (ER), LAMP1 (late endosomes and lysosomes), UNC93B1–Flag and TLR7–HA across fractions was measured by immunoblot. Data are representative of three independent experiments. b, Workflow for isolation of phagosomes from RAW macrophages and purification of UNC93B1–Flag complexes from phagosome lysates.
Extended Data Fig. 5 Syntenin-1 and syntenin-2 inhibit TLR7 signalling.
a, Syntenin-1 is selectively recruited to UNC93B1 after TLR7 stimulation, but not TLR3 stimulation. Syntenin-1 binding to UNC93B1 was measured by Flag immunoprecipitation followed by immunoblot for syntenin-1 from RAW macrophage lines stimulated with R848 (0.5 μg ml−1) or poly(I:C) (10 µg ml−1) for the indicated times. Levels of syntenin-1 and UNC93B1–Flag in cell lysates are also shown. b, Syntenin-1 associates selectively with the TLR7–UNC93B1 complex, but not with TLR9. Syntenin-1 binding to TLR7–HA or TLR9–HA was measured by haemagglutinin immunoprecipitation followed by immunoblot for syntenin-1 from indicated RAW macrophage lines stimulated with R848 (0.5 μg ml−1) or CpG-B (0.5 µM) for the indicated times. Levels of syntenin-1 and TLR7–HA or TLR9–HA in cell lysates are also shown. c, NF-κB activation in HEK293T cells transiently expressing syntenin-1 and stimulated with TNF (10 ng ml−1). d, NF-κB activation in HEK293T cells transiently expressing TLR7 and increasing amounts of syntenin-2. Cells were stimulated with R848 (50 ng ml−1) for 16 h before collection. Data in c and d were measured using a dual luciferase reporter assay, normalized to Renilla expression and expressed as RLUs. Data are mean ± s.d., n = 3 biological replicates. *P < 0.05, **P < 0.01 and ***P < 0.001, one-way ANOVA followed by a Tukey’s post-test (95% confidence interval). All data are representative of at least three independent experiments.
Extended Data Fig. 6 UNC93B1(K333R) confers enhanced TLR7 signalling without affecting TLR9 and TLR3.
Flow cytometry analysis showing the percentage of TNF-positive cells, measured by intracellular cytokine staining of UNC93B1-deficient RAW macrophages expressing the indicated alleles after stimulation with CpG (25 nM), R848 (8 ng ml−1), poly(I:C) (20 µg ml−1), or LPS (10 ng ml−1). Shaded histograms show unstimulated controls. Data are representative of three independent experiments.
Extended Data Fig. 7 Serine phosphorylation in the C-terminal tail of UNC93B1 restricts TLR7 signalling.
a, b, Validation of the anti-phospho-UNC93B1 polyclonal UNC93B1 antibody. a, Immunoblots demonstrating the specificity of the phospho-specific antibodies generated against Ser547 and Ser550 in the UNC93B1 C-tail. Varying quantities of synthesized peptides corresponding to the UNC93B1 C-terminal regulatory region with (P-UNC93B1-C) and without (NP-UNC93B1-C) phosphorylated Ser547 and Ser550 were dropped onto membrane and probed with rabbit phospho-specific, affinity-purified polyclonal anti-UNC93B1 IgG. Data are representative of two independent experiments. b, Phospho-specific polyclonal antibodies detect both phosphorylated Ser547 and Ser550. UNC93B1 was isolated from UNC93B1-deficient RAW macrophages expressing UNC93B1 mutants S547A, S550A or S547A/S550A by Flag immunoprecipitation followed by immunoblot with phospho-specific polyclonal antibodies. Data are representative of at least three independent experiments. c, Intracellular cytokine staining of TNF in macrophage lines expressing the indicated UNC93B1 alleles and stimulated with CpG (10 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (20 µg ml−1), or LPS (10 ng ml−1). Grey histograms are unstimulated controls. d, TNF production, measured by ELISA, from the indicated RAW macrophage lines after stimulation for 8 h with LPS (50 ng ml−1). Data are mean ± s.d., n = 3 biological replicates. Data are representative of three independent experiments. e, Levels of phosphorylated p38 and JNK, as measured by immunoblot, in lysates of the indicated RAW macrophage cells stimulated with R848 (50 ng ml−1). Data are representative of two independent experiments.
Extended Data Fig. 8 Genetic variation in the human UNC93B1 C-terminal regulatory region increases TLR7 responses.
NF-κB activation in human HEK293T cells transiently expressing TLR7 or TLR5 and the indicated human UNC93B1 alleles was measured using a dual luciferase reporter assay. Cells were stimulated with R848 (10 ng ml−1) or flagellin (2 ng ml−1) for 16 h before collection. Data are normalized to Renilla expression and expressed as RLUs. Data are mean ± s.d., n = 3 biological replicates. *P < 0.05, **P < 0.01 and ***P < 0.001, one-way ANOVA followed by a Tukey’s post-test (95% confidence interval). Data are representative of three independent experiments.
Extended Data Fig. 9 UNC93B1PKP knock-in mice develop systemic inflammation.
a. CRISPR–Cas9 strategy to generate UNC93B1PKP knock-in mice. Green line indicates the guide sequence. Red bases indicate the edited codons. A representative sequencing trace of genomic DNA from an edited founder mouse is shown. b, Flow cytometry analysis of the indicated immune cell populations in 6–8-week-old Unc93b1WT/WT, Unc93b1PKP/WT and Unc93b1PKP/PKP mice. Frequencies of dendritic cells (CD11b+CD11c+MHCIIhigh) and inflammatory monocytes (CD11b+Ly6c+Ly6Gneg) in lymph nodes are shown. Data points were pooled from four independent experiments. P values determined by unpaired two-tailed Student’s t-test. c, Unc93b1PKP/PKP mice exhibit signs of emergency granulopoiesis in their bone marrow compartment. Flow cytometry analysis of bone marrow from 6–8-week-old Unc93b1WT/WT, Unc93b1PKP/WT and Unc93b1PKP/PKP mice. Gates representing LSK (CD45+CD3ε−CD19−Ly6c−Ly6G−Sca-1highc-Kithigh) and Sca-1highc-Kit− cells (CD45+CD3ε−CD19−Ly6c−Ly6G−Sca-1highc-Kit−) are indicated and compiled frequencies of Sca-1highc-Kit− cells are shown on the right. Data are mean ± s.d., n = 3 biological replicates. P value determined by unpaired two-tailed Student’s t-test. d, Representative staining, corresponding to compiled results shown in Fig. 4c, of anti-nuclear antibodies (ANA) using sera from the indicated mouse ages and genotypes. e, f, Flow cytometry analysis showing percentage of TNF-positive cells, measured by intracellular cytokine staining of BMMs and BMDCs derived from the indicated mice after stimulation with CpG-B (150 nM), R848 (10 ng ml−1), ssRNA (1 µg ml−1), poly(I:C) (10 µg ml−1), or LPS (10 ng ml−1). Shaded histograms are unstimulated controls. g, TNF production by BMDCs derived from the indicated mice after stimulation for 8 h with R848, CpG-B (150 nM) or LPS (50 ng ml−1). h, TNF production by BMMs from the indicated mice after stimulation for 8 h with CpG-B (500 nM), LPS (50 ng ml−1), or increasing concentrations of R848. Data in g and h are mean ± s.d., n = 3 biological replicates. P values determined by unpaired two-tailed Student’s t-test. i, B cells from Unc93b1PKP/PKP mice show enhanced proliferation in response to TLR7 stimulation. Proliferation of CFSE-labelled B cells after 3 days stimulation with R848 (8 ng ml−1) or LPS (1.6 µg ml−1) was measured by FACS, pre-gating on live CD19+ cells. The proliferation index is determined by dividing the geometric mean fluorescent intensity (gMFI) of the fluorescent dye CSFE of the unstimulated control by the gMFI of CSFE of the stimulated sample (CSFEunstim/CFSEsample). Data are mean ± s.d., n = 5 mice per group pooled together from three independent experiments. P values determined by unpaired two-tailed Student’s t-test. j, Immunoprecipitation of MyD88 from BMMs from the indicated mice after stimulation with R848 (500 ng ml−1), followed by immunoblot for IRAK2. Input levels of MyD88 and IRAK2 in whole-cell lysates are also shown. k, UNC93B1 protein levels in BMMs from indicated mouse genotypes, measured by immunoblot with polyclonal antibodies against endogenous UNC93B1. All data are representative of three independent experiments, unless otherwise noted.
Extended Data Fig. 10 Gating strategies.
Representative gating strategies for marginal zone B cells, activated T cells, dendritic cells, inflammatory monocytes, emergency granulopoiesis in bone marrow and B cell proliferation in splenocyte cultures are shown. These strategies were used for the data presented in Fig. 4 and Extended Data Fig. 9.
Supplementary information
Supplementary Figure 1
This file contains the uncropped gel images for main figures and extended data figures. Separate gels were run for detection of each protein. When loading or normalization controls were used, these samples were run on separate gels.
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Majer, O., Liu, B., Kreuk, L.S.M. et al. UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity. Nature 575, 366–370 (2019). https://doi.org/10.1038/s41586-019-1612-6
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DOI: https://doi.org/10.1038/s41586-019-1612-6
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