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The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease

Abstract

TRAF1 is a signaling adaptor known for its role in tumor necrosis factor receptor-induced cell survival. Here we show that monocytes from healthy human subjects with a rheumatoid arthritis–associated single-nucleotide polymorphism (SNP) in the TRAF1 gene express less TRAF1 protein but greater amounts of inflammatory cytokines in response to lipopolysaccharide (LPS). The TRAF1 MATH domain binds directly to three components of the linear ubiquitination (LUBAC) complex, SHARPIN, HOIP and HOIL-1, to interfere with the recruitment and linear ubiquitination of NEMO. This results in decreased NF-κB activation and cytokine production, independently of tumor necrosis factor. Consistent with this, Traf1−/− mice show increased susceptibility to LPS-induced septic shock. These findings reveal an unexpected role for TRAF1 in negatively regulating Toll-like receptor signaling, providing a mechanistic explanation for the increased inflammation seen with a disease-associated TRAF1 SNP.

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Figure 1: Activated T cells from human subjects with an RA-associated SNP express less TRAF1 and produce less cytokines.
Figure 2: Monocytes from human PBMCs with an RA-associated SNP express lower TRAF1 protein and produce more inflammatory cytokines in response to LPS.
Figure 3: Effect of TRAF1 SNP on cytokine expression in PBMCs stimulated with LPS plus anti-CD3 and on expression of A versus G alleles in heterozygous subjects.
Figure 4: TRAF1 regulates LPS-induced inflammatory responses in the human monocytic cell line THP-1, independently of TNF signaling.
Figure 5: TRAF1 associates with the LUBAC complex by directly binding HOIP and HOIL-1 via its MATH domain.
Figure 6: TRAF1 limits linear ubiquitination of NEMO to control NF-κB activation.
Figure 7: LPS-stimulated monocytes with the RA-associated SNP exhibit enhanced linear ubiquitination.
Figure 8: Traf1−/− mice are more susceptible to endotoxin-induced septic shock.

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Acknowledgements

We thank Genentech for providing linear ubiquitin–specific antibody, B. Ghumman and J. Warzyszynska for technical assistance, L.E. Wagar for collecting and genotyping some of the PBMC samples, G. Ehrhardt for providing the control-GST construct and D. Philpott, S. Girardin and M. Hayden for suggestions and comments. Supported by grants MOP-74492 and FDN-143250 from the Canadian Institutes of Health Research (CIHR) to T.H.W. T.H.W. is supported by the Sanofi Pasteur Chair in Human Immunology at the University of Toronto. D.L.C. was supported by a doctoral award from CIHR and the Canadian Association for HIV research.

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A.A.A.-S. and T.H.W. conceived, designed and interpreted experiments and wrote the manuscript, with input from M.I.E. and D.L.C. A.A.A.-S. conducted experiments with the help of M.I.E., A.M. and D.L.C. E.K. provided anti-human TRAF1 monoclonal antibody.

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Correspondence to Tania H Watts.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gating strategy for flow cytometric analysis of T cells and frequency of T cell subsets

(a) Gating strategy for the flow cytometry analysis performed on T cells from healthy human subjects’ PBMCs for TRAF1 measurements in T cells, as in Fig. 1a. (b) Representative histograms showing TRAF1 expression in stimulated and unstimulated T cells from PBMCs of AA, AG and AG donors. (c) control knockdown (shCTRL) or TRAF1 knockdown (shTRAF1) RAJI cells were used to confirm specificity of TRAF1 stain. (d) Frequency of the various subsets of T cells from donor PBMCs with the AA, AG or GG genotype were measured by flow cytometry using the antibodies listed in the methods section. n = 26, 36, 15 human subjects of each genotype AA, AG, GG respectively. Results are representative from 2 similar experiments. Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test.

Supplementary Figure 2 TRAF1 protein levels in unstimulated T cells and gating strategies for T cell cytokines and monocytes

(a) TRAF1 levels were measured by flow cytometry using a PE labeled anti-hTRAF1 antibody in unstimulated T cells from donor PBMCs with the AA, AG or GG genotypes. Each symbol represents one donor; n = 26, 36, 15 human donors of each genotype AA, AG, GG, respectively. Results are representative of 2 similar experiments. Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test. * p <0.05 (b) Gating strategy for intracellular flow cytometry analysis performed on T cells from healthy human donor PBMCs for cytokine measurements, as in Fig. 1b. (c) Gating strategy for the flow cytometry analysis performed on purified monocytes from healthy human donor PBMCs, as in Fig. 2.

Supplementary Figure 3 Flow cytometric measurements of TRAF1 and cytokines from human PBMC monocytes

(a) Purified monocytes from healthy human subjects’ PBMCs were treated or not with LPS (100 ng/ml for 2, 4 or 24 hrs), and TRAF1 levels were measured by flow cytometry using a PE-labeled anti-hTRAF1 antibody. (a) Graphical representation of the mean fluorescence intensities (dMFI = MFI of sample – MFI of FMO) of TRAF1 in purified monocytes of donors with the CC, CT and TT genotypes of the TRAF1 SNP rs2900180. Each symbol represents one donor; n = 29, 19, 10 human donors of each genotype CC, CT, TT, respectively. (b-d) Purified monocytes from healthy human donor PBMCs were treated or not with LPS (100 ng/ml for 4, 6 or 8 hrs), and percentage of cytokine producing cells was assessed by flow cytometry using an (b) IL-1β antibody, IL-6 antibody (c) or TNF antibody (d). (b-d) Each symbol represents one donor; n = 22, 22, 14 human donors of each genotype AA, AG, GG, respectively. Results are representative of 2 similar experiments. (a-d) Statistical analysis was performed using a non-parametric (Mann Whitney) unpaired t-test. * p < 0.05 and ** p < 0.01

Supplementary Figure 4 shTRAF1 THP-1 cells have enhanced NF-κB activation and express higher levels of inflammatory cytokines than shCTRL cells after TLR or NOD ligand treatment

(a) shCTRL (shC) and shTRAF1 (shT) THP-1 cells were stimulated with Pam3CSK4 (P3C; 10 ng/ml), Flagellin (Fla; 0.5 μg/ml), Resiquimod (R848; 10 ng/ml), Poly(I:C) (I:C; 10 μg/ml), L18-muramyl dipeptide (MDP; 10 ng/ml) or LPS (100 ng/ml) for the indicated times and whole cells extracts (WCE) were immunoblotted for phospho-IκB-α (p-IκB-α). Blots are representative of two independent experiments (b) Gene expression of TNF, CCL5, IL6 and p40 (IL12) was evaluated by real-time PCR (Q-PCR) in 100 ng/ml LPS stimulated shCTRL or shTRAF1 THP-1 cells for the indicated times as in Fig. 4e. Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. * p < 0.05, ** p < 0.01 and *** p < 0.001 (c) TNF levels were measured by Enzyme-linked immunosorbent assay (ELISA) in supernatants of anti-CD3/anti-CD28 stimulated T cells from 23 human donors PBMCs with or without co-treatment with 1 μg/ml Adalimumab (Ada) for 48 hrs. Graphs show the mean ± SEM. Statistical analysis was performed using a paired t-test. *** p < 0.001

Supplementary Figure 5 Status of NEMO ubiquitination following LPS and TNF stimulation

(a) IP of endogenous Lys48 ubiquitin chains (K48 Ub) or Lys63 ubiquitin chains (K63 Ub) followed by immunoblotting for NEMO as in Fig. 6 panel c, with whole cell lysates, as a loading control, shown to the right. (b) Analysis of TNFR1-associated NEMO ubiquitination status and TNFR1 immunoprecipitates in Flag-TNF-treated shCTRL and shTRAF1 THP-1 cells. Lysates were first immunoprecipitated with anti-Flag (IP1: Flag), dissociated and re-immunoprecipitated with anti-linear ubiquitin antibody (IP2: Linear Ub) or anti-K63 ubiquitin antibody (IP2: K63 Ub), and blotted for TNFR1. Whole cell lysates are shown in the bottom panel. (c) NF-κB firefly luciferase reporter was cotransfected into HEK293 FT cells with control Renilla luciferase and LUBAC (HOIP + HOIL-1). Some cells were additionally transfected with increasing amounts of TRAF1 or with 125 ng of TRAF2 expression vectors, as indicated. Luciferase activity was measured after 48 h. Graphs show the mean ± standard deviation of three independent experiments (d) Gene expression of TRAF1, TNF, CCL5 and p40 (IL12 β) was evaluated by real-time PCR (Q-PCR) in 100 ng/ml LPS stimulated shCTRL or shTRAF1 THP-1 cells (treated with non-targeting siRNA (siCTRL) or HOIP targeting siRNA (siHOIP) for 48 hrs) for the indicated times as in panel a. Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. (e) LUBAC inhibition by gliotoxin reverses the enhanced NF-κB activation in shTRAF1 THP-1 cells. WCE from control knockdown (shCTRL) and TRAF1 knockdown (shTRAF1) THP-1 cells treated with LPS (100 ng/ml) for the indicated times were immunoblotted for IκB-α (left panels) or as a loading control GAPDH (right panels). Some shCTRL THP-1 cells were pretreated (15’) with 1 μM Gliotoxin. Blots are representative of 3 independent experiments.

Supplementary Figure 6 Proposed mechanism for TRAF1 role in TLR and TNFR1 signaling

TRAF1 limits linear ubiquitination of NEMO in both the TLR and TNFR1 signaling pathways, but these effects in TNFR1 signaling are counterbalanced by the role of TRAF1 in recruitment of cIAP1 to enhance K63-linked polyubiquitination of NEMO.

Supplementary Figure 7 Macrophages from Traf1–/– mice are hyper-responsive to TLR stimulation

(a) d5 bone marrow macrophages (BMMs) from C57BL/6NCrl (WT) mice were stimulated with 10 ng/ml LPS for the indicated times, and gene expression was assessed by reverse-transcriptase PCR (RT-PCR). (b) BMMs from WT (left) or Traf1-/- (right) littermate mice were stimulated with 10 ng/ml LPS for the indicated times, and whole cells extracts (WCE) were immunoblotted for total IκB-α (IκB-α), phospho-ERK1/2 (p-ERK1/2) and as a loading control GAPDH. Blots are representative of at least three independent experiments. (c) Gene expression of Ifn-β, Tnf, Il1-β and Traf1 was evaluated by real-time PCR (Q-PCR) in LPS stimulated BMMs for the indicated times as in panel a (10ng/ml LPS). (d) d5 bone marrow macrophages (BMMs) from WT or Traf1-/- littermate mice were stimulated with 100 ng/ml or 1 μg/ml LPS for the indicated times, and gene expression of Ifn-β, Ip-10, Tnf, Il-1β and Kc was evaluated by real-time PCR (Q-PCR). (c-d) Results were normalized to GAPDH and reported as relative fold change w.r.t. untreated control. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. * p < 0.05, ** p < 0.01 and *** p < 0.001

Supplementary Figure 8 Traf1–/– macrophages produce higher levels of cytokines than WT

(a) Tnf levels were measured by Enzyme-linked immunosorbent assay (ELISA) in supernatants from WT (Traf1+/+) or KO (Traf1-/-) BMMs treated with 10 ng/ml, 100 ng/ml or 1 μg/ml LPS for the indicated times. Graphs show the mean ± standard deviation of three independent experiments, which were compared using a 2 way ANOVA with multiple comparisons. *** p < 0.001 (b) The levels of Tnf, Il6 and Ccl5 secreted into the supernatants of LPS stimulated WT (Traf1+/+) heterozygous (Traf1+/-) or KO (Traf1-/-) BMMs were measured using Bio-plex cytokine assay. Graphs show the mean ± standard deviation of three independent experiments, which were compared using unpaired t-test; * p < 0.05 and *** p < 0.001

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Abdul-Sater, A., Edilova, M., Clouthier, D. et al. The signaling adaptor TRAF1 negatively regulates Toll-like receptor signaling and this underlies its role in rheumatic disease. Nat Immunol 18, 26–35 (2017). https://doi.org/10.1038/ni.3618

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