DHA (D7439, 150 mg), lipopolysaccharide (LPS), phorbol myristate acetate (PMA) and MTX hydrate (M8407) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Complete Freund’s adjuvant (CFA) (7001, 10 ml), incomplete Freund’s adjuvant (IFA) (7002, 10 ml), and bovine type II collagen (20021, 10 ml) were purchased from Chondrex, Inc. (Redmond, WA, United States). ST486 cell line (70014502, CRL-1647), RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from ATCC Co., Ltd. (United States). AffiniPure goat anti-human IgM (109-005-129) was obtained from Jackson ImmunoResearch Co., Ltd. (PA, United States). Fluo-4 AM (F312) was purchased from Dojindo Co., Ltd. (Japan). The primary antibody against CD32B (ab45143, EP888Y) was purchased from Abcam Co., Ltd. (Cambridge, United Kingdom). The primary antibody against GAPDH (10494-1-AP) was obtained from Proteintech Group, Inc. (United States). The primary antibodies against Lyn (C13F9) (2796), SHP-1 (C14H6) (3759), and phospho-CD19 (Tyr531) (3571) were purchased from CST Co., Ltd. (United States).

The animals used for this experiment were male DBA/1J mice (Vital River Laboratory Animal Technologies Co. Ltd. Beijing, China) that were 8 weeks old. The mice were housed in a laminar flow cabinet with a 12 h light/dark cycle and maintained on specific pathogen-free (SPF) laboratory chow and water ad libitum. All the experimental studies were strictly in accordance with Guangzhou University of Chinese Medicine Animal Ethics Committee guidelines for the rational use of animals.

After 1 week of adaptation, 24 DBA/1J mice were randomly divided into two groups: control group (n = 6), arthritis-induced group (n = 18). To prepare mouse CIA model, 0.1 ml of emulsion of bovine type II collagen and CFA (1:1, v/v) was injected intradermally into the tail base of mice in arthritis-induced group on day 0, as the primary immunization. A booster injection of 0.1 ml of emulsion of bovine type II collagen and IFA (1:1, v/v) was administered intradermally into the back on day 21, as the secondary immunization (Zhang et al., 2013; Miyoshi and Liu, 2018). Mice in control group injected normal saline at the same location and frequency as arthritis-induced group. The clinical symptoms were recorded weekly since the secondary immunization, with the arthritis index scores according to the following criteria: 0 = normal; 1 = erythema and mild swelling; 2 = erythema and swelling extending to ankle joints and one or two toes; 3 = erythema and swelling extending to metatarsal joints and more than two toes; and 4 = ankylosing deformity with joint swelling. The scores from each paw were added to obtain a cumulative score between 0 and 16. In addition, swelling in the paw was measured using digital calipers, and body weight was recorded during the course.

After the secondary immunization on day 21, mice in the arthritis-induced group were randomly divided into three groups (n = 6 per group): model group, DHA group (20 mg/kg, daily), MTX group (2 mg/kg, every 3 days). DHA and MTX was dissolved in corn oil and administrated by oral gavage from day 21 to day 49. Control and model mice were orally given an equal volume of corn oil in parallel.

After 4 weeks of drug administration, the right hind knee and ankle of each mouse was withdrawn and scanned by Skyscan 1176 micro-CT scanner (Bruker micro-CT, Kontich, Belgium). The scanning was carried out using following settings: voltage, 80 kV; source current, 88 μA; pixel size 4 μm. Two and three-dimensional images were generated using Data-viewer and CTvol softwares (Bruker micro-CT, Kontich, Belgium) respectively. The bone mineral density (BMD) and bone volume/tissue volume (BV/TV, %) were measured using CT Analyser program (Bruker micro-CT, Kontich, Belgium).

ST486 cells were maintained in RPMI-1640 medium in a humidified chamber with 37°C at 5% CO₂. ST486 cells that expressed wild-type human FcγRIIb or the I232T loss-of-function mutant were constructed. Murine stem cell virus (pMSCV) puro plasmids contained human FcγRIIb-I232T or FcγRIIb-WT were used as templates, and inserted into lentiviral expression vector (pLVX) via the EcoRI and MluI restriction enzyme sites after PCR amplification. Then the expression vector constructs of human FcγRIIb-WT or FcγRIIb-I232T were transfected to ST486 by electroporation. The stable sublines of ST486 cells expressing equivalent levels of human FcγRIIb-I232T or FcγRIIb-WT were sorted by at least two rounds of fluorescence-activated cell sorting. Quantitative real-time PCR (qRT-PCR) assays were performed to verify the transfection. The methods for RNA purification and qRT-PCR will be described below. The specific primer sets in Supplementary Table S1. After the transfection, transfected cells (Mut group) were incubated with or without different concentrations of DHA or MTX to perform the following studies.

ELISA kit (Beijing 4A Biotech Co., Ltd., Beijing, China) were used to determine the cytokine production under DHA treatment. Transfected cells (Mut group) were divided into six groups: NC (without DHA and LPS), LPS (150 ng/ml), DHA (250 ng/ml), DHA (500 ng/ml), DHA (1000 ng/ml), and MTX (500 ng/ml, positive control group). Twenty-4 hours after transfection, the cells (1 × 10⁵/ml) were seeded in 24-well plates and treated with different concentrations of DHA or MTX (500 ng/ml) for 48 h, then PMA was added and incubated at 37°C with 5% CO₂ for 48 h. The supernatant was then discarded, LPS (150 ng/ml) was added to each group except the NC group, and the cells were incubated overnight at 37°C with 5% CO₂. The culture supernatants were collected, and the amount of TNF-α released from the cells was detected according to the manufacturer’s instructions. IL-6 cytokine assays were carried out using the same method.

qRT-PCR was performed to analyze the expression of TNF-α and IL-6 in ST486 cells treated with DHA or MTX. Total RNA was isolated with TRIzol (Invitrogen, United States) and reverse transcribed into cDNA using a Prime Script RT Reagent kit (Takara Biotechnology, Dalian, China) according to the manufacturer’s protocol. qRT-PCR was completed after 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min. The relative fold-changes of gene transcriptions were determined by 2^−△△Ct method with the specific primer sets in Supplementary Table S1.

The experiment was divided into six groups, which involved FcγRIIb-WT (control group), FcγRIIb-Mut (model group), MTX (500 ng/ml, positive control group), DHA (250 ng/ml), DHA (500 ng/ml) and DHA (1000 ng/ml). 10 μL of CCK-8 solution was added after culturing with different concentrations of DHA or MTX for 48 h. Then cells were incubated under the same conditions for another 2 h. The absorbance at 490 nm was measured by a microplate reader (iMark, Thermo Fisher Scientific, United States) to evaluate cell viability.

Annexin V apoptosis detection kit (BD Biosciences, United States) was used to measure cell apoptosis. After treating with different concentration of DHA or MTX as described above, the transfected cells were harvested and incubated with 7-AAD and Annexin V antibody following the manufacturer’s instructions. Then, cells were immediately analyzed by a FACSCalibur flow cytometer.

Multiple signal transduction pathways can affect changes in intracellular Ca²⁺ concentration, and thus the detection of intracellular Ca²⁺ changes can help to understand the initiation, enhancement or inhibition of cellular functions. In this study, we used flow cytometry to monitor the dynamic changes of intracellular Ca²⁺ concentration in order to dynamically observe the degree of B-cell activation and thus verify whether DHA can inhibit the B-cell activation. After treating with different concentration of DHA or MTX as described above, the transfected cells (1.5 × 10⁶/ml) were incubated in RPMI 1640 medium containing Fluo-4 AM (5 μΜ) for 1 h in a humidified chamber with 37°C at 5% CO₂ to load with Fluo-4 AM. Then it was washed 3 times and detected by a flow cytometer constantly. The cells were stimulated with anti-human IgM (20 μg/ml) at 50 s, CaCl₂ was added back to a final concentration of 2 mM at 400 s, and intracellular Ca²⁺ flux was measured for 800 s.

After treating with different concentration of DHA or MTX as described above, cells were stimulated with anti-human IgM (20 μg/ml) for 15 min. Protein lysates obtained from equal numbers of ST486 cells were separated by SDS-PAGE and then transferred to 0.4 μm PVDF membranes via electroblotting. After being blocked by skimmed milk for 1 h, membranes were incubated with antibodies against GAPDH, phospho-CD19, CD32B, Lyn, and SHP-1 overnight at 4°C. Then the membranes were incubated with the corresponding secondary antibodies at room temperature for 1 h. GAPDH was used as an internal control. Immunoreactive bands were visualized through Enhanced Chemiluminescence method and recorded in gel documentation system.

All experiments were repeated at least three times, and all data are presented as the mean ± SEM. Statistics were analyzed using SPSS (version 20.0, IBM, Armonk, NY) while graphs were drawn by GraphPad Prism software (version 7.0). Significant differences between multiple groups were calculated using one-way ANOVA followed by Tukey’s test. Values of p < 0.05 were considered significant.

DHA and MTX treatment did not cause weight loss in CIA mice (Figure 1A). Mice in DHA (20 mg/kg, daily), MTX (2 mg/kg, every 3 days) group showed significant reduction in paw oedema between day 28 and day 49 compared with mice in model group (p < 0.05, Figures 1B,C). Collagen challenge induced arthritis in mice, as evidenced by red swelling in the paw (Figure 1D). Micro-CT analysis showed that DHA or MTX treatment ameliorated the bone loss, as BMD and BV/TV in both DHA and MTX group were significantly higher than those in model group (p < 0.05, Figures 1D–H). Besides, no significant difference was observed between DHA and MTX groups in any of the above analyses.

As seen in Figure 2A, after the plasmid was digested, an obvious band could be seen around 8000bp, and there were 23bp bases between the two digestion sites, so it could not be detected. After the plasmids were digested by EcoRI + MluI, obvious bands could be seen at 8000 bp and 1000 bp attachment, indicating that the 951 bp sequence of the target gene had been inserted. The sequencing results showed correct and the plasmids were confirmed. Figure 2B shows the sequence maps of wild-type and variant FcγRIIb encoding membrane penetrating proteins, showing that variant FcγRIIb has a variation at site 232. As shown in Figure 2C, successful plasmid transfection was verified by qRT-PCR, and our results showed that when compared to the WT group, the relative expression of FcγRIIb was significantly decreased in both of the Con and Mut groups (p < 0.0001 and p < 0.01, respectively).

Accumulated evidence reveals that certain proinflammatory cytokines contribute to the pathogenic factors supporting the proliferation and activation of B cells (Carvalho et al., 2011; Gottenberg et al., 2012; Lee et al., 2017). Therefore, we assessed the role of DHA in the regulation of inflammatory cytokine production by LPS-stimulated ST486 cells. As shown in Figures 3A,B, compared with the LPS group, the protein expression levels of TNF-α and IL-6 were significantly lower in both the DHA and MTX-treated groups, except for the protein expression level of TNF-α in the DHA (250 ng/ml) group. In addition, as seen in Figures 3C,D, we also found that IL-6 and TNF-α mRNA levels were decreased in the DHA-treated group compared to the LPS group, while the difference compared with the MTX group was not statistically significant.

CCK-8 is used to detect the proliferation of ST486 cells and to evaluated the influence of DHA on the proliferation of ST486 cells. Figure 4A showed that when compared with those in the Mut group, the proliferation of ST486 in the WT, MTX and different dose of DHA goroup were all significantly decreased (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). Besides, when compared with the MTX group, the proliferation of ST486 in the different dose of DHA group were all significantly increased (p < 0.01, p < 0.05, and p < 0.05, respectively).

The apoptotic cells were detected by flow cytometry. As shown in Figures 4B,C, when compared with that in the Mut group, the number of apoptotic cells in the WT, MTX and DHA-treated groups were all significantly increased (p < 0.0001, p < 0.0001, p < 0.0001, p < 0.0001, and p < 0.0001, respectively). Additionally, when compared with the MTX group, the numbers of apoptotic cells in the DHA-treated (250 ng/ml) was significantly decreased (p < 0.0001), the numbers of apoptotic cells in the WT group was increased (p < 0.0001).

Previous studies have showed that FcγRIIb selectively dephosphorylated CD19 in consequence with an inhibition of BCR signaling (Koncz et al., 1998). Therefore, the western blot analysis was used to investigate whether the effect of DHA is correlated with the CD19 pathway. When compared to the cells in the WT group, the intracellular Ca²⁺ level in the Mut group was remarkably increased after 100 s. However, when compared with the Mut group, the calcium concentration decreased more rapidly in DHA and MTX group after 200 s (Figure 5B). Besides, as illustrated in Figure 5F, when compared with the Mut group, the protein expression levels of phosphorylated CD19 were all downregulated in the WT, DHA-treated (500 and 1000 ng/ml) and MTX groups (p < 0.05, p < 0.05, p < 0.05, and p < 0.01, respectively). These data all indicated that DHA inhibited B cell activation.

Previous studies have shown that FcγRIIb signal pathway plays an important role in B cell activation (Hippen et al., 1997), so we further study the effect of DHA on this signal pathway. (Figure 5A). The results showed that when compared to those in the Mut group, there were a significant increased in CD32B, Lyn and SHP-1 expression in the WT group (p < 0.05, p < 0.05, and p < 0.01, respectively, Figures 5C–E). Additionally, the protein expression of CD32B in DHA (500 and 1000 ng/ml) groups were much higher than that in the Mut group (p < 0.05 and p < 0.05, respectively). Besides, the Lyn expression in the DHA (1000 ng/ml) and MTX groups were significantly higher than that in the Mut group (p < 0.05 and p < 0.05, respectively). Correspondingly, when compared with the Mut group, consistent results for the SHP-1expression in the DHA (1000 ng/ml) and MTX groups (p < 0.01 and p < 0.05, respectively). In all, these data suggested that DHA impeded B cells activation induced by IgM through the modulation of the FcγRIIb/Lyn/SHP-1 pathway.