Animals

Seven-week-old male DBA/1 J mice (Orient Bio, Gyeonggi-do, Korea) were purchased from Orient Bio Inc. (South Korea). The animals were maintained under specific pathogen-free conditions in an animal facility with controlled humidity (55 ± 5%), light (12 h/12 h light/dark), and temperature (22 ± 1 °C). The air in the facility passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mice chow and water ad libitum. All experimental procedures were provided in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals and the Guidelines and Policies for Rodent experiment provided by the IACUC (Institutional Animal Care and Use Committee) in school of medicine, The Catholic University of Korea. To generate SMILE transgenic mice, a pcDNA3.0 vector was constructed containing a cytomegalovirus promoter. The SMILE fragment was synthesized by GenScript Corporation (USA), with codon optimization for expression in mammalian cells. The open reading frame originated in mice. Transgenic mice overexpressing SMILE were generated on a C57BL/6 background by transgene microinjection and maintained at Macrogen Inc. (South Korea). SMILE transgenic founder mice were mated to C57BL/6 mice and were crossed for 10 generations. The presence of the transgene in the founders was confirmed by polymerase chain reaction (PCR) using genomic DNA extracted from the tail. All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: 2021-0010-01, 2020-0306-02).

Induction and assessment of collagen induced arthritis (CIA) and SMILE administration

To induce CIA, 0.1 mL of emulsion containing 100 μg of bovine type II collagen (CII) and Freund’s complete adjuvant (Chondrex, WA, USA) and Freund’s complete adjuvant (Chondrex) was injected intradermal into the base of the tail as a primary immunization. Two weeks later, a booster injection of 100 μg CII dissolved and emulsified 1:1 with water, a booster Freund’s incomplete adjuvant (Difco, MI, USA) was administered into the tail. At 1 week after collagen-induced arthritis (CIA) induction, SMILE mock control vector was administered by electroporation; weekly hydrodynamic injection was then performed for 10 weeks, as described previously. SMILE overexpression experiments, SMILE cDNA fragment was cloned into the pcDNA3.0 vector between EcoRI and KpnI. Escherichia coli containing the SMILE overexpression vector were incubated in Luria–Bertani high salt broth (Duchefa Biochemie, Netherlands) for 16 h at 37 °C and 160 rpm. The cells were harvested by centrifugation, the SMILE overexpression vector purified using a NucleoBond Xtra Maxi EF Kit (Macherey–Nagel, Germany). Mice were intravenously injected with 100 μg of SMILE overexpression vector or control vector in 1 mL of saline.

Induction and assessment of collagen antibody-induced arthritis (CAIA)

Eight-week-old male C57BL/6 mice and SMILE transgenic mice were used for the induction of CAIA. Anti-collagen antibodies 2 mg (Chondrex) were injected intravenously in to 2 groups of mice, and 3 days later, 50 μg of LPS was administered intraperitoneally. Mice were monitored and evaluated daily for arthritis.

Clinical assessment of arthritis

The severity of arthritis was evaluated by three independent observers. The mice were observed twice weekly or daily to determine the onset and severity of joint inflammation for up to 7 weeks after the primary immunization. The severity of arthritis was assessed on a scale of 0–4, based on the following criteria: 0 = no edema or swelling 1 = slight edema, with erythema with erythema limited to the foot or ankle; 2 = slight edema, with erythema from the ankle to the tarsal bone; 3 = moderate edema, with erythema from the ankle to the tarsal bone; and 4 = severe edema, with erythema from the ankle to the entire leg. The arthritis score of each mouse was calculated as the sum of the scores of the four limbs; the highest possible arthritis score for each mouse was 16. The mean arthritis index was used to compare the scores of the control and experimental groups [29].

Histological analysis

Mouse joint tissues were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA), decalcified in a histological decalcifying solution (Calci-Clear Rapid; National Diagnostics, Atlanta, GA, USA), and embedded in paraffin wax for histological analyses. Sections (7 mm) were prepared and stained with hematoxylin (YD Diagnostics, Yongin, Korea), eosin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan), and safranin O (Sigma-Aldrich) [30].

Immunohistopathological analysis of arthritis

Joint tissues were incubated overnight at 4 °C with primary antibodies against SMILE (Abcam, Cambridge, UK), B-cell activating factor receptor (BAFF-R) (Abcam), pAMPK (Abcam), mTOR (Cell Signaling, MA, USA), pSTAT3 ser727 (Abcam), Pstat3 Tyr705 (Abcam), IL-1β (Abcam), IL-6 (Abcam), IL-17(Abcam). Subsequently, samples were incubated with a biotinylated streptavidin–peroxidase complex for 1 h, and the signals were developed using chromogen 3,3′- diaminobenzidine (Thermo Scientific, Rockford, IL, USA). The sections were examined under a photomicroscope (Olympus, Tokyo, Japan). The number of positive cells in high-power digital images (magnification, ×400) was counted using Adobe Photoshop software (Adobe, San Jose, CA, USA). Stained cells were counted independently by three observers, and the mean values were evaluated.

Immunofluorescence imaging

Th17 (CD4, IL-17), Treg (CD4, CD25, Foxp3), SMILE, AMPK, mTOR, pSTAT3 705, pSTAT3 727 and germinal center markers (CD3, B220, PNA) expression levels were analyzed by confocal microscopy. Tissue Sects. (7 μm thick) were fixed in methanol-acetone and stained with phycoerythrin (PE)-conjugated anti-CD4 (Biolegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti CD4 (BD Bioscience, San Diego, CA, USA), PE-conjugated anti-interleukin-17 (eBioscience, San Diego, CA, USA), allophycocyanin (APC)-conjugated anti-CD25 (Biolegend), PE-conjugated anti-Foxp3 (Thermo Fisher Scientific, Waltham, MA, USA), PE-conjugated anti-phosphorylated Stat3 (Tyr 705, Ser 727, BD Bioscience), APC-conjugated anti-B220 (eBioscience), Alexa 594-conjugated anti-PNA (Thermo Fisher Scientific), SMILE (Abcam, Cambridge, UK), AMPK (Abcam) and mTOR (Cell Signaling). After overnight incubation at 4℃, the stained sections were analyzed on a Zeiss confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany) at 200× magnification and Zen Blue edition software.

Western blotting

Cells were lysed in RIPA lysis and extraction buffer containing Halt protease inhibitor cocktail (Thermo Scientific, USA). Lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to membranes (GE Healthcare, USA). The membranes were incubated with antibodies against SMILE, phosphorylated AMPK (Cell Signaling, MA, USA), SMILE, GAPDH (Abcam, Cambridge, UK). Hybridized bands were detected by enhanced chemiluminescence (Thermo Scientific, USA) on X-ray film (AGFA, Belgium). Western blotting was performed using the SNAP i.e. Protein Detection System.

Isolation and stimulation of splenocytes and culture

Splenocytes were prepared from the spleens of Collagen induced arthritis mice. Splenocytes were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 5% fetal bovine serum (Gibco, Grand Island, NY, USA). Splenocytes were stimulated with or Curcumin (Sigma-Aldrich) 25 μg and LPS (Sigma-Aldrich) 100 ng/mL for 12 h and 72 h; they were then subjected to western blot analysis and flow cytometry, respectively.

Flow cytometry

The spleens were removed from the mice and single cells were isolated and stained using fluorescently conjugated antibodies against Follicular B and Marginal B cells; APC-conjugated anti-B220 (eBioscience), Percp/Cy5.5-conjugated CD23 (Biolegned) and Pacific Blue-conjugated CD21 (Biolegend), Mature B and Immature B cells; APC-conjugated anti-B220 (eBioscience), FITC-conjugated anti IgD (Thermo Fisher Scientific) and PE-conjugated anti-IgM (Thermo Fisher Scientific), regulatory B cells; FITC-conjugated anti-CD5 (Invitrogen), PE-conjugated CD1d (eBioscience) and PE/Cy7-conjugated anti-CD19 (BD Bioscience), Plasma B; ACP-conjugated anti-B220 (eBioscience) and PE-conjugated anti-CD 138 (BD Bioscience), Germinal center B; APC-conjugated anti-B220 (eBioscience) and Alexa Flour 488-conjugated anti-GL7 (Biolegend), IL-17 secreting B; PE/Cy7-conjugated anti-CD19 (BD Bioscience) and PE-conjugated anti-interleukin-17 (eBioscience) and BAFF-R; PE/Cy7-conjugated anti-CD19 (BD Bioscience) and PE-conjugated anti-CD268 (Biolegend). Prior to intracellular staining, cells were stimulated for 4 h with phorbol 12-myristate 13-acetate (PMA) (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop™ (BD Biosciences, USA). Intracellular staining was performed using a BD Cytofix/Cytoperm Plus Fixation/Permeabilization Kit and BD Golgistop Kit (BD Biosciences, USA). The transcription factor Foxp3 was stained using a Foxp3/Transcription Factor Staining Kit (eBioscience, USA) according to the manufacturer’s instructions. Flow cytometry was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, USA).

Measurement of immunoglobulin

Serum was gathered when sacrificed for concentration of Total IgG, IgG1 and IgG2a, and the serum was stored at – 70 °C until further use. The levels of the Total IgG, IgG1 and IgG2a antibodies in the serum were measured using ELISA (Bethyl Laboratories, Montgomery, TX, USA).

Statistical analysis

Statistical analysis was performed using Prism9 (GraphPad Software, USA). Normally distributed continuous data were analyzed by parametric Student’s t-test. Differences in means among groups were subjected to analysis of variance followed by Bonferroni post hoc test. Values are means ± standard error of the mean (SEM). A value of P < 0.05 was indicative of statistical significance.