TMEM203 is a binding partner and regulator of STING-mediated inflammatory signaling in macrophages

Significance Activators of interferons have received a great deal of interest in recent decades, both due to the central role they play in host defense against a range of pathogens, as well as the now well-recognized importance of dysregulated interferon activation/signaling in the pathogenesis of a number of highly prevalent and hard-to-treat diseases, such as systemic lupus erythematosus (SLE). Therefore, novel regulators of interferon activation are being sought as they may provide better targets to treat these diseases. We report the discovery of TMEM203 as an SLE-associated gene and a regulator of ligand-dependent activation of interferon production via STING. Thus, our work could form the basis of a novel therapeutic strategy for the treatment of interferonopathies, including SLE.


Generation of CRISPR/Cas9 Tmem203 Knockout RAW 264.7 cells
Four suitable sgRNA oligos were designed and used in the generation of pGL3-U6 sgRNA-Puromycin expression vectors. Transfection of RAW 264.7 with pGL3-U6 sgRNA expression vectors was carried out using Lipofectamine 2000. Colonies were selected for establishment of a stable cell lines along with CRISPR-Cas 9 control group using selective medium containing Puromycin (2 μg/ml) and Blasticidin (10 μg/ml).

Generation of Tmem203 Stably Expressing RAW 264.7 cells
Mouse Tmem203 cDNA was cloned into the pcDNA 3.1 vector then transfected into RAW 264.7 cells using Lipofectamine 2000 (Invitrogen) and 500μg/ml G418 was used for the selection of stably expressing Tmem203 cells.

Standard and quantitative real-time PCR
For analysis of mRNA levels following treatment, cells were washed with PBS and harvested for total RNA extraction using standard protocols. Complementary DNA was made from 500 ng total RNA (Biorad iScript cDNA synthetic kit) for gene expression analysis. Real-time quantitative PCR was performed using Primerdesign SYBR green mastermix in 10μl reaction volume in triplicate wells and quantified in BioRad CFX384 Touch TM real-time PCR cycler. Primers for amplification of human genes are listed in Supplementary   Table 2.

Site-directed Mutagenesis and molecular cloning
Single-amino acid mutation was performed using standard protocols. Fidelity of the constructs was confirmed by Sanger sequencing. Primers used for mutagenesis are listed in Supplementary Table 2.

Protein complementary assay
To determine protein interaction using split Venus protein complementary assay (PCA), HEK293T cells were transfected. Post transfection, cells were harvested in FACS buffer and assessed by flow cytometry. Cell viability was determined by TO-PRO-3 (Thermo Fisher).
For split Renilla PCA, HEK293T cells were transfected. Post transfection, Renilla signal was analysed using Nano-Glo® Luciferase Assay System (Promega). Cells were stimulated with STING ligands for the indicated period and Renilla activities measured. For STIM1-TMEM203 competition with STING, increasing dose of pcDNA3.1-STIM1-his was transfected, as stated in the figure legend. Cells were also stained with Hoechst33324 (ThermoFisher) to indicate cell number for normalisation. Cells were maintained at 37℃ throughout the experiment.

Immunostaining and confocal microscopy:
HeLa cells were transiently transfected with Tmem203-mCherry for 48 hours using metafectene (Biontex) and treated with 1μg/ml LPS for 30 and 60 minutes as indicated. Cells were fixed and stained with an anti-LAMP1 antibody, (H5G11, SC-18821, Santa Cruz) and localisation was visualised using Alexa Fluor 488 conjugated anti-mouse secondary antibody (Molecular Probes). Bone-marrow isolated from WT and Tmem203 -/-C57BL/6 mice were differentiated into BMDMs for 5 days using standard protocols and were then treated with 10 µg/ml 2'-3' cGAMP for 3 hours. Cells were fixed, permeabilised with 0.1% (v/v) Triton X-100 and stained with anti-STING antibody (Abcam ab181125) and ER or Lysosome Cytopainter staining kit (Abcam) as indicated. STING localisation in the ER or lysosomes was visualised using Alexa Fluor 647 conjugated anti-mouse secondary antibody (Abcam).
For live cell imaging of Tmem203-Sting organelle localisation, HeLa cells were transfected with V2_Tmem203 (WT or mutant) and V1_Sting (WT or mutant) for 18-24 hours using Lipofectamine 3000. Post transfection, samples were stained for 10 mins with ER or lysosome Cytopainter staining kit (Abcam). Images were acquired by confocal microscopy on a Zeiss LSM 510 META with 40X inverted water-lens (Molecular Probes). Images were analysed using Fiji software. ROIs were selected around the organelles to calculate the number of pixels of the organelle, Sting-Tmem203 fluorescence and co-localised signals. The percentage of localisation was calculated as the ratio of co-localised pixels to the number of pixels occupied by the organelle.
RAW 264.7 macrophages stably expressing Tmem203 or empty vector were grown on coverslips and transfected with 1 µg 3'3'-cGAMP for 6h, along with controls. Cells were fixed with 3.7% (v/v) formaldehyde for 15 minutes at 37°C. Fixed and permeabilised cells were incubated with rabbit anti-IRF3 antibody (Cell Signaling Technology) before incubation with Alexa Fluor 546-conjugated donkey anti-rabbit IgG. Confocal fluorescence images were captured on a Zeiss LSM510 META microscope.
Human MDMs were permeabilised with 0.1% (v/v) Triton X-100 then washed and blocked followed by incubation with mouse anti-human CD68 (Dako) then with AlexaFluor 488-conjugated goat anti-mouse secondary antibodies. Nuclei were visualised with DAPI. Cells were imaged on a LEICA AFI6000 Time-Lapse microscope at 10X magnification.

Western-blotting and immunoprecipitation analysis
Post treatment, cells were harvested in RIPA buffer (Sigma Aldrich) and phosphatase inhibitors (Roche) and analysed using standard western blotting protocols.

Statistical analysis
Data was analysed using GraphPad Prism (8.0) and is presented as mean ± SEM, unless stated otherwise.

Fig. S1. Alignment of TMEM203 orthologues:
Homologues of the mouse TMEM203 protein were identified in GenBank and aligned using the Clustal W algorithm. Four transmembrane regions (TM1-4) were predicted by TMpred (6).  B. Human monocyte-derived macrophages express CD68. CD14 positive monocytes were differentiated into macrophages with M-CSF incubation and purity was examined by blotting with anti-human CD68 and detected with Alexa Fluor® 488 conjugated goat anti-human. Scale bar = 100 µm.
Mean IFN-β mRNA levels (relative to β-actin) ±SEM are plotted from 3 biological replicates for each MDM culture.

S4. A-C. Venus PCA Tmem203 and Sting co-expression does not induce ER stress in HEK293 T cells.
Co-transfection of Tmem203 (A) and Sting (B) Venus PCA constructs resulted in high expression of both genes but a relatively small increase of XBP1 mRNA splicing (C).