Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel

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1 Supplementary Figures 1-8 Crucial role for human Toll-like receptor 4 in the development of contact allergy to nickel Marc Schmidt 1,2, Badrinarayanan Raghavan 1,2, Verena Müller 1,2, Thomas Vogl 3, György Fejer 4, Sandrine Tchaptchet 4, Simone Keck 4, Christoph Kalis 4, Peter Nielsen 4, Chris Galanos 4, Johannes Roth 3, Arne Skerra 5, Stefan F. Martin 6, Marina A. Freudenberg 4, and Matthias Goebeler 1,2 1 Department of Dermatology, University Hospital Mannheim, University of Heidelberg, D Mannheim, Germany, and 2 Department of Dermatology, University of Giessen, D Giessen, Germany 3 Institute of Immunology, University of Münster, D Münster, Germany 4 Max-Planck-Institute for Immunobiology, D-7918 Freiburg, Germany 5 Munich Center for Integrated Protein Science and Chair for Biological Chemistry, Technical University Munich, D-8535 Freising-Weihenstephan, Germany 6 Allergy Research Group, Department of Dermatology, University Medical Center Freiburg, D-7914 Freiburg, Germany Nature Immunology: doi:1.138/ni.1919

abrogates - induced expression of the NF-κB target gene CCL2.

Flow cytometric analysis of intracellular CCL2 expression

5 mm), IL-1β (1 U ml -1 ) or TNF (2 ng ml -1 ) for 15 h.

2 Supplementary Figure 1. Expression of a dominant negative mutant of IRAK1, IRAK1-DD, abrogates - induced expression of the NF-κB target gene CCL2. vector IRAK1-DD IL-1β Events CCL-2 TNF Flow cytometric analysis of intracellular CCL2 expression after stimulation of ECs with (1.5 mm), IL-1β (1 U ml -1 ) or TNF (2 ng ml -1 ) for 15 h. Filled profiles represent CCL2 expression, empty profiles isotype controls. Cells have been gated on co-transfected green fluorescent protein to control for efficient transfection. Data are representative of two independent experiments. Nature Immunology: doi:1.138/ni.1919

3 Supplementary Figure 2. Proinflammatory activation by requires. a IL-8 mrna (fold stimulation) HEK293 WT b IL-8 (pg/ml) htlr1-2 HEK293 -hmd2 5 4 NS htlr2 htlr3 - htlr1-2-6 htlr7 htlr8 hmd2 * medium Polymyxin B htlr9 (a) qrt-pcr of IL-8 mrna expression in HEK293 cells transfected with the indicated htlr receptors and stimulated with medium () or for 3 h. Functionality of the respective htlr genes was controlled using appropriate agonists (not shown). (b) Quantification of IL-8 in the supernatants of HEK293 cells expressing and hmd2 upon 8 h stimulation with medium (), or in the absence or presence of 5 µg ml -1 polymyxin B sulphate. Data represent means of two to four experiments ± s.d. * p <.1 (unpaired t-test). Nature Immunology: doi:1.138/ni.1919

4 Supplementary Figure 3. The failure of mtlr4 to respond to is not due to inefficient expression. HA SSC 1 2 Events (% of max) IL-8 mtlr4 Flow cytometry analysis of intracellular IL-8 and HA staining in hmd2-expressing HEK293 cells transfected with HA- or HA-mTLR4 after treatment with (1.5 mm) for 8 h. The indicated cell population in the dot blots represents the successfully transfected HA-positive live cell population (HA + ), used as gate for the IL-8 quantifications (histograms). Histograms are identical to those presented in Fig. 3d. Percentages of total HA positivity in the live population and of IL-8 positivity in the gated HA + population are indicated. Data are representative of three independent experiments. Nature Immunology: doi:1.138/ni.1919

5 Supplementary Figure 4. Non-conserved histidines H456 and H458 of are required for activation by. a b c IL-8 MFI (fold) IL-8 (fold induction compared to ) IL-8 mrna (fold stimulation) HEK293 hmd2 NS ** 25 NS (H456A) HEK293 hmd HUVEC IgG 2a control ** (H456A) NS NS (H458A) * (H458A) α- (HTA125) ** (H456A- H458A) (H456A- H458A) HA Tubulin Vector (H456A) (H458A) (H456A- H458A) (a,b) HEK293 stably expressing hmd2 were transfected with HA-tagged TLR4 constructs containing the indicated point mutations or empty expression vector as control. Cells were exposed to medium (), or (Salmonella minnesota R595; 1 µg ml -1 ) for 8 h and subsequently processed for intracellular flow cytometry (a) or ELISA (b) to detect IL-8. (a) Flow cytometric quantification of averaged isotype-corrected median fluorescence intensities (MFIs) from three independent experiments ± s.d. Cells have been gated for positive transfection by anti-ha co-staining. (b) ELISA analysis of IL-8 release in response to stimulation with. Data are shown in relation to induction by (arbitrarily set to 1) and represent averages ± s.d of three independent experiments. Successful expression of the transgenes was confirmed by Western blot for the HA-tag (b). (c) qrt-pcr for IL-8 expression in HUVEC stimulated with medium (), or in the presence of 2 µg ml -1 of the α- antibody HTA125 or a corresponding IgG 2a isotype control. Data are presented as mean ± s.d of four independent experiments. *p <.5, **p <.1; NS = not significant (unpaired t-test). Nature Immunology: doi:1.138/ni.1919

6 Supplementary Figure 5. Structural modeling of Ni2+ interaction with. TLR4 TLR4* MD2 Ni2+ Ni2+ MD2* Modeling of the (Ni2+)2-()2 complex in ribbon representation (side view) with the putative positions of the bound Ni2+ molecules indicated. The model is based on the crystal structure of the -hmd2 complex (PDB entry 3FXI) with omitted. Molecular modeling and graphics preparation were performed using PyMOL software. Nature Immunology: doi:1.138/ni.1919

7 Supplementary Figure 6. Expression of TLR4 in transgenic Tlr4 -/- -TG and Tlr4 -/- mtlr4-tg mice and human peripheral blood monocytes. a Events (% of Max) human monocytes (CD14 hi ) Tlr4 -/- -TG mouse monocytes (F4/8 hi ) 15 mouse BM-derived macrophages (Live) 6 b Tlr4 -/- mtlr4-tg Tlr4 -/- -TG 4 3 splenic DCs (CD14c + ) Events (% of Max) TLR4 4 3 splenic macrophages (F4/8 + ) (a) Flow cytometry analysis of surface expression on human peripheral blood monocytes (CD14 hi ), murine peripheral blood monocytes (F4/8 hi ) and purified bone marrow (BM)-derived macrophages from Tlr4 -/- -TG mice. Numbers in the displayed histogram overlays represent ratios of geometric mean fluorescence intensity () obtained for the indicated α- stained population (black lines) and its corresponding IgG control (grey lines). Population gates are indicated in brackets. (b) Flow cytometric analysis of TLR4 surface expression on splenic DCs (CD11c + ) and splenic macrophages (F4/8 + ) from Tlr4 -/- -TG and Tlr4 -/- mtlr4-tg mice. Cells were studied for expression of the respective TLR4 transgene using an appropriate species-specific TLR4 antibody (black lines). Alternatively, cells were stained with an antibody directed against TLR4 of the non-matching species (i.e. with α-mtlr4 in case of the Tlr4 -/- -TG cells and α- in case of the Tlr4 -/- mtlr4-tg derived cells; grey lines) as background control. Numbers display background-related geometric means of fluorescence for the corresponding TG lines. Gates are indicated in brackets. Data are representative of two or three independent experiments. Nature Immunology: doi:1.138/ni.1919

a Supplementary Figure 7. -induced activation of mouse macrophages.

28 28 IL-1 (pg/ml) 21 14 7 IL-1 (pg/ml) 21 14 7 b Tlr4 -/- -TG Tlr4 -/- mtlr4-tg 3 6 12 3 6 12 Time (min) p-p38 c p38 3 Tlr4 -/-

background were exposed to 1 mm,.1 µg ml -1 (Salmonella minnesota R595) or medium as indicated.

(b,c) Western blot analysis of -induced p38 protein phosphorylation (b) or IRF3 protein phosphorylation (c) in macrophages

8 a Supplementary Figure 7. -induced activation of mouse macrophages. Tlr4 -/- -TG Tlr4 -/- mtlr4-tg IL-1β (pg/ml) IL-1β (pg/ml) IL-6 (pg/ml) IL-6 (pg/ml) IL-1 (pg/ml) IL-1 (pg/ml) b Tlr4 -/- -TG Tlr4 -/- mtlr4-tg Time (min) p-p38 c p38 3 Tlr4 -/- -TG Tlr4 -/- mtlr4-tg 3 IFN-α/ß (U/ml) Time (min) Time (min) p-irf3 p-irf3 Tubulin Tubulin IFN-α/ß (U/ml) 22.5 (a-c) Bone marrow-derived macrophages obtained from transgenic mice expressing either (left) or mtlr4 (right) on a C57BL/1ScCr background were exposed to 1 mm,.1 µg ml -1 (Salmonella minnesota R595) or medium as indicated. Thereafter, synthesis of cytokines IL-1β, IL-6, IL-1 (a) or IFN-α/β (c) was determined by ELISA. Data represent means ± s.d. of three independent experiments. (b,c) Western blot analysis of -induced p38 protein phosphorylation (b) or IRF3 protein phosphorylation (c) in macrophages derived from Tlr4 -/- -TG or Tlr4 -/- mtlr4-tg mice. Cells were stimulated for the indicated time intervals. Equal loading of lanes was controlled by immunostaining of p38 or tubulin, respectively. Data are representative of two independent experiments. Nature Immunology: doi:1.138/ni

9 Supplementary Figure 8. Proinflammatory signaling pathways activated by Ni2+. For induction of contact allergy to Ni2+ two signals are required: an antigen-specific, T-lymphocyte-activating signal ( signal 1") and a proinflammatory signal ( signal 2"). The latter signal is sensed by -hmd2 resulting in the activation of NF-κB, p38 and IRF3 and subsequent expression of cytokines, adhesion molecules and type I interferons. Nature Immunology: doi:1.138/ni.1919

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