MRL/lpr mice were purchased from SLC Inc. (Shizuoka, Japan). They were housed in groups of five in polycarbonate cages in a specific-pathogen-free environment. The mice had access to standard mouse chow (Ralston Purina Co., St. Louis, MO, USA) and water ad libitum. Eight-week-old MRL/lpr mice were orally administered 5 mg/kg Tac (MedChemExpress, Monmouth Junction, NJ, USA) and/or 50 mg/kg LA (CNS Pharm Korea Co., Ltd., Jincheon, Korea), daily for 8 weeks. The LA were heat-killed at 80°C for 30 min prior to administration. All experimental procedures were approved by the Animal Research Ethics Committee of the Catholic University of Korea (approval number: 2020-0151-04).
Caecal DNA extraction, PCR amplification and sequencing
Total DNA was extracted using the Maxwell® RSC PureFood GMO and Authentication Kit (Promega, USA), in accordance with the manufacturer’s instruction. PCR amplification was performed using fusion primers targeting from V3 to V4 regions of the 16S rRNA gene with the extracted DNA. For bacterial amplification, fusion primers of 341F (5’-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTC-AGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3’; underlining sequence indicates the target region primer) and 805R (5’- CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGG-AGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3’). The Fusion primers are constructed in the following order which is P5 (P7) graft binding, i5 (i7) index, Nextera consensus, Sequencing adaptor, and Target region sequence. The amplifications were carried out under the following conditions: initial denaturation at 95 °C for 3min, followed by 25 cycles of denaturation at 95 °C for 30 sec, primer annealing at 55 °C for 30 sec, and extension at 72 °C for 30 sec, with a final elongation at 72 °C for 5 min. The PCR product was confirmed by using 1% agarose gel electrophoresis and visualized under a Gel Doc system (BioRad, Hercules, CA, USA). The amplified products were purified with the CleanPCR (CleanNA). Equal concentrations of purified products were pooled together and removed short fragments (non-target products) with CleanPCR (CleanNA). The quality and product size were assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled and the sequencing was carried out at Chunlab, Inc. (Seoul, Korea), with Illumina MiSeq Sequencing system (Illumina, USA) according to the manufacturer’s instructions.
Caecal microbiome data analysis pipeline
Processing raw reads started with quality check and filtering of low quality (<Q25) reads by Trimmomatic ver. 0.32 . After QC pass, paired-end sequence data were merged together using fastq_mergepairs command of VSEARCH version 2.13.4  with default parameters. Primers were then trimmed with the alignment algorithm of Myers & Miller  at a similarity cut off of 0.8. Non-specific amplicons that do not encode 16S rRNA were detected by nhmmer  in HMMER software package ver. 3.2.1 with hmm profiles. Unique reads were extracted and redundant reads were clustered with the unique reads by derep_fulllength command of VSEARCH . The EzBioCloud 16S rRNA database  was used for taxonomic assignment using usearch_global command of VSEARCH  followed by more precise pairwise alignment . Chimeric reads were filtered on reads with <97% similarity by reference based chimeric detection using UCHIME algorithm  and the non-chimeric 16S rRNA database from EzBioCloud. After chimeric filtering, reads that are not identified to the species level (with <97% similarity) in the EzBioCloud database were compiled and cluster_fast command  was used to perform de-novo clustering to generate additional OTUs. Finally, OTUs with single reads (singletons) are omitted from further analysis. The secondary analysis which includes diversity calculation and biomarker discovery was conducted in EzBioCloud 16S-based MTP, which is a Chunlab, Inc (Seoul, South Korea) bioinformatics cloud platform.
Splenocytes and peripheral blood were immunostained with surface eFluor780-conjugated fixable viability dye (eBioscience, San Diego, CA, USA), Pacific Blue-conjugated anti-CD90.2 (Biolegend), peridinin-chlorophyll-protein-cyanine5.5-conjugated anti-CD4 (eBioscience), phycoerythrin (PE)-conjugated anti-CD8 (Biolegend), and allophycocyanin (APC)-conjugated anti-CD2 (Biolegend). After fixation and permeabilization, cells were stained with fluorescein isothiocyanate (FITC)-conjugated IL-17 (eBioscience) and PE-conjugated Foxp3 (eBioscience). For intracellular staining, cells were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate and 250 ng/mL ionomycin (Sigma, St. Louis, MO, USA) for 4 h in the presence of GolgiStop (BD Biosciences, San Diego, CA, USA). The data were analysed using FlowJo software (Tree Star, Ashland, OR, USA).
Histological analyses were performed to quantify spleen and kidney inflammation. Kidney tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Spleen tissue cryosections were fixed in methanol-acetone. Kidney and spleen sections were stained with haematoxylin and eosin, examined under a photomicroscope (Olympus, Tokyo, Japan), and scored .
Immunohistochemistry was performed using a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA, USA). Briefly, tissue sections were incubated overnight at 4°C with a primary antibody against SIGNR3 (R&D Systems, Minneapolis, MN, USA), followed by a biotinylated secondary antibody, and then reacted with a streptavidin-peroxidase complex for 1 h. 3,3’-Diaminobenzidine (Dako, Carpinteria, CA, USA) was added as a chromogen, and the samples were visualised using a microscope (Olympus).
Tissue cryosections (7-µm-thick) were fixed in methanol-acetone and stained with FITC-conjugated anti-CD4, APC-conjugated anti-CD25, PE-conjugated anti-IL-17, and -Foxp3 (eBioscience). After incubation at 4°C overnight, the stained sections were analysed using a Zeiss microscope (LSM 510 Meta; Carl Zeiss, Oberkochen, Germany) at 200× magnification.
Blood was collected from the orbital sinus, and serum samples were stored at –20°C until use. Serum levels of anti-dsDNA antibodies were measured using poly-L-lysine, dsDNA-cellulose (Sigma), and mouse IgG detection antibody (Bethyl Laboratories, Montgomery, TX, USA). IgG2a levels were measured using ELISA kits (Bethyl Laboratories). The levels of IL-10 and IL-17 in the cultured supernatants from MRL/lpr splenocytes were measured using sandwich ELISA (R&D Systems). Absorbances were determined using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).
Urine albumin and creatinine assays
Urine albumin and creatinine concentrations were measured using a mouse albumin ELISA assay (Bethyl Laboratories) and a creatinine assay (R&D systems), respectively, according to the manufacturer’s instructions.
Real-time polymerase chain reaction (PCR)
mRNA was extracted using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) as per the manufacturer’s instructions. Complementary DNA was synthesised using a Super Script reverse transcription system (TaKaRa, Shiga, Japan). A Light-Cycler 2.0 Instrument (software v4.0; Roche Diagnostics, Indianapolis, IN, USA) was used for the PCR amplification. All reactions were performed using the LightCycler FastStart DNA Master SYBR Green I Mix (TaKaRa) following the manufacturer’s instructions. The following primers were used: SIGNR3, 5’-TCA-AGA-GTT-TGG-CAG-AGT-ATA-CG-3’ (sense) and 5’-TTG-TTC-TGA-ACC-TCT-GAG-CTG-3’ (antisense); IDO, 5’-GAC-GGA-CTG-AGA-GGA-CAC-AG-3’ (sense) and 5’-GGC-AGC-ACC-TTT-CGA-ACA-TC-3’ (antisense); PD-L1, 5’-AAA-GTC-AAT-GCC-CCA-TAC-CG-3’ (sense) and 5’-TTC-TCT-TCC-CAC-TCA-CGG-GT-3’ (antisense); IL-10, 5’-GGC-CCA-GAA-ATC-AAG-GAG-CA-3’ (sense) and 5’-AGA-AAT-CGA-TGA-CAG-CGC-CT-3’ (antisense); β-actin, 5’-GAA-ATC-GTG-CGT-GAC-ATC-AAA-G-3’ (sense) and 5’-TGT-AGT-TTC-ATG-GAT-GCC-ACA-G-3’ (antisense). All mRNA levels were normalised to β-actin.
siRNA for SIGNR3 was purchased from Cosmo Genetech (Seoul, Korea). Before transfection, murine non-T-cells were cultured with LPS (100 ng/mL; Sigma) from Escherichia coli O111:B4. The next day, the cells were transfected using the Amaxa 4D-nucleofector X unit with a primary cell kit, as per the manufacturer’s recommendations (Lonza, Cologne, Germany).
PBMC isolation and stimulation
PBMCs of healthy volunteers and SLE patients were prepared from heparinised blood using a standard Ficoll-Paque density gradient centrifugation (GE Healthcare Biosciences, Uppsala, Sweden). Cells were cultured in RPMI-1640 medium (Gibco BRL, Carlsbad, CA, USA) and stimulated with anti-CD3 (0.5 µg/mL) for 3 days. All procedures were approved by the ethics committee of Seoul St. Mary’s Hospital (Seoul, Republic of Korea).
Statistical analyses were performed using Prism software (v5.0; GraphPad Software Inc., San Diego, CA, USA). Differences between groups were evaluated using t-tests (two-tailed) for two groups and one-way analysis of variance for three or more groups. P < 0.05 was considered statistically significant.