Reagents and chemicals
Inosine, hypoxanthine, xanthine, guanine and uric acid were obtained from MedChemExpress (Shanghai, China) and were prepared as 1000 ppm methanol stocks. NaOH 10 mM was used as necessary to increase solubility. MMF was purchased from Tokyo Chemical Industry (Shanghai, China) and acetonitrile was purchased from Macklin (Shanghai, China).
The study group included 34 NMOSD patients who had received MMF therapy (0.5 g MMF orally, twice a day for long term therapy) at the Third Affiliated Hospital of Sun Yat-sen University (Guangzhou, China). The primary inclusion criteria were (1) clinical diagnosis of NMOSD and seropositive for AQP4-IgG; (2) patients aged 15-65 and (3) an Kurtzke Expanded Disability Status Scale (EDSS) score ≤ 8.0. Patients who met any of the following key criteria were excluded: (1) treatment with antibiotics or probiotics within the last 3 months; (2) emergency treatment such as plasmapheresis or intravenous methylprednisolone for acute recurrence within the last 1 month; (3) Concurrent disease with diabetes mellitus, systemic autoimmune diseases or other neurological diseases.
Stool and serum samples were taken from the patient for 0.5, 1 and 2 h after receiving MMF in the early morning. Clinical data that was collected included follow-up time, relapses and EDSS score. The 34 participants were divided into two groups; patients that had decreased CNS symptoms and decreased EDSS scores were assigned to the effect group (EG) and patients whose symptoms were not altered were treated as the non-effect group (NEG) .
This study was approved by the ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (02-016-01) and all patients signed informed consent.
Microbiome 16S rRNA gene sequencing, alignment and analysis
Microbial DNA was extracted from fecal samples of NMOSD patients using the E.Z.N.A. Stool DNA Kit (Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer’s protocol. Extracted DNA was qualitatively and quantitatively analyzed by gel electrophoresis and ultraviolet spectroscopy before sequencing. Thereafter, bacterial 16S rDNA V3–V4 regions were amplified using primers 341F and 806R (Illumina, San Diego, CA, USA). The resulting amplicons were sequenced using the Illumina NovaSeq 6000 platform (Guangdong Longsee Biomedical, Foshan, China).
Stool sample processing and HPLC-UV–MS/MS analysis were performed as previously described with minor modifications . In brief, chromatographic separations were conducted using a Shimadzu LC-30AD series HPLC system (Shimadzu, Kyoto, Japan) with an Frulic N column (AZYP, LLC, Arlington, USA). Analytes were eluted at a flow rate of 0.4 mL/min using a gradient of phase A (0.2% formic acid) and phase B (0.2% formic acid in acetonitrile) as follows: 0–0.5 min, 80% B; 0.5–4.5 min, 50% B; 4.5–8 min, 80% B. The eluates were monitored by UV at 254 nm and MS detection in parallel. The injection volume as 5 μL, and the column temperature was maintained at 23°C. MS detection was performed using an AB Sciex Triple Quad 5500 (AB Sciex, Toronto, Canada). The MS source parameters were set as follows: drying gas temperature, 300°C; gas flow rate, 10 L/min; sheath gas temperature, 350°C; sheath gas flow rate, 12 L/min; nebulizer, 45 psig; capillary voltage, 3500 V; and nozzle voltage, 500 V. Nitrogen was used as the drying and collision gas. Detection was conducted in positive mode using multiple reaction monitoring (MRM).
Female C57BL/6 mice aged 6 weeks were used in all experiments. Animals were maintained in Laboratory animal center of South China Agricultural University (Guangzhou, China). Mice were housed in sterilized, filtertop cages under specific pathogen–free conditions and fed control diets for a minimum of 1 week before commencing experiments. Vancomycin was administered in drinking water for 14 days in advance to deplete part of gut microbiome and then fed with 0.563% (w/w) MMF for 7 consecutive days and designated as Van-MMF combined intervention group (VM). The MMF group were fed MMF for 7 days without vancomycin treatment and controls received normal feed and drinking water (Fig. 3A). MMF was supplemented at 0.563% (w/w) into the diets for experimental groups. All of the animal experiments adhered to the institution’s guidelines for animal use and the Guide for the Care and use of Laboratory Animals at laboratory animal center of South China Agricultural University (Guangzhou, China). Whole blood was collected from anesthetized animals by cardiac puncture. Serum were isolated by centrifugation at 3000 rpm for 10 min and stored at −80°C.
Metabolomes of mouse serum were profiled using a GC-MS-based untargeted metabolomic procedure. The samples were added into an Eppendorf tube with 200 μL extractant solution (acetonitrile: methanol =1: 1, including internal standard of isotope-labeled winged peptide) and vortexed for 30 s and then incubated at -40°C for 1 h. The samples were centrifuged at 12 000 rpm for 15 min at 4°C and the resulting supernatant was transferred to a clean glass vial for analysis as described previously . The quality control (QC) sample was the combination of the supernatants from all of the samples.
LC-MS analyses were performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 µm) coupled to a Q Exactive HAF Mass Spectrometer (Orbitrap MS, Thermo Fisher, Pittsburg, PA, USA). Mobile phase A was consisted of 25 mmol/L ammonium acetate and 25 ammonia hydroxide in water (pH = 9.75). Mobile phase B was acetonitrile. The injection volume was 3 μL. The QE HFX mass spectrometer was used for its ability to acquire MS/MS spectra on information-dependent acquisition (IDA) mode in the control of the Xcalibur 4.0.27 acquisition software (Thermo Fisher). In this mode, the acquisition software continuously evaluates the full scan MS spectrum. The electrospray ionization (ESI) source conditions were set as follows: sheath gas flow rate 30 arb, aux gas flow rate 25 arb, capillary temperature 350°C, full MS resolution as 60, 000, MS/MS resolution as 7,500, collision energy as 10/30/60 in NCE mode and spray voltage as 4.0 kV (positive) or 3.6 kV (negative).
Data processing and statistics
Raw data were converted into the mzXML format using ProteoWizard and processed for peak detection, extraction, alignment and integration with an in-house program that was developed using R and based on XCMS (Smith et al., 2006). An in-house MS2 database (Biotree DB, Shanghai, China) was applied for metabolite annotations with a mass tolerance of 10 ppm. The cutoff for annotation was set at 0.3 and 6068 peaks were detected and 279 metabolites remained after noise reduction relative to the standard deviation in serum samples and the internal standard was used for data normalization. The final dataset contained the peak number, sample name and normalized peak area and was imported into the SIMCA 15.0.2 software package (Sartorius Stedim Data Analytics (Umea, Sweden). Data were scaled and log- transformed and further analyzed as previously described .
The data were expressed as the mean ± standard deviation (SD). A nonparametric T-test was performed for comparison of the two groups using Prism v8.0 software (GraphPad, San Diego, CA, USA). A p value of 0.05 was considered to be statistically significant.