Isolation and identification of EFL2 (C₃₈H₄₂O₉, Figure 1A) were performed as described before (Zhang et al., 2018). The purity of up to over 98% was determined by HPLC. EFL2 was dissolved in DMSO as a stock solution. The stock solution was diluted (1:100) with sterile PBS supplemented with 3% tween 20 before administrated to mice (the final concentration of DMSO is 1%).

K/BxN serum was collected from K/BxN mice at 8 weeks of age, pooled, aliquoted and stored at -20 °C. K/BxN serum-transfer arthritis (STA) model was induced by intraperitoneal (i.p.) application of 150 μL of serum at day 0. Clinical score for arthritis severity was assessed using a 0–4 point scale for each paw at various time points (0–16 total score) as described previously (Luo et al., 2011). 8-week old male C57BL/6 mice were purchased from Dashuo Animal Center (China, Chengdu), maintained at 25°C, and exposed to 12-h light and dark cycles. Mice were fed with standard laboratory chow and water with 1 week of acclimatization. Naive mice were used as the control group. Except the control mice, all mice were i. p. injected with 150 μL of serum, then randomly divided into the following groups and treated with different compounds daily for the consecutive 8 days: 1) STA group; 2) Solvent control group, mice were gavaged with the solvent; 3) Dexamethasone (DEX) group, mice were gavaged with 30 mg/kg DEX; 4) EFL2 group 1, mice were gavaged with 40 mg/kg EFL2; 5) EFL2 group 2, mice were intraperitoneally injected with 15 mg/kg EFL2; 6) EFL2 group 3, the control mice were intraperitoneally injected with 40 mg/kg EFL2. In addition, the control mice were intraperitoneally injected with 40 mg/kg EFL2. All animal experiments were conducted in accordance with ethical regulation for animal care and use in China.

Prior to the pharmacokinetic study, 12 male C57BL/6 mice were fasted for 12 h with free access to water, and then treated by intraperitoneal injection of EFL2 at a dose of 40 mg/kg. About 50 μL blood samples were collected via the oculi chorioideae vein at 0.5, 1, 2, 4, 8, 12, 24, 72, 96, and 168 h, respectively. After centrifugation at 5,000 rpm for 10 min, plasma samples were obtained and frozen at −80°C until analysis. The pharmacokinetic parameters of EFL2 were calculated using WinNolin 4.0.1 software.

20 μL plasma samples were spiked with 340 μL acetonitrile by vortex mixing for 30 s. The mixture was then centrifuged at 10,000 rpm for 5 min at 4°C to separate the precipitated protein. An aliquot of 50 μL supernatant was transferred to a fresh tube and spiked with 450 μL mobile phase (acetonitrile containing 0.1% formic acid). After centrifugation at 13,000 rpm for 5 min, 3 μL supernatant was injected into Agilent 6,495 Triple Quadrupole LC/MS/MS system for each time. Then, the concentration of EFL2 was determined.

An Agilent 1,200 series UPLC system (Agilent Technologies, Waldbronn, Germany) equipped with an 6,495 quadruple mass spectrometer (Agilent Technologies, Santa Clara, CA, United States) were used for pharmacokinetic study. The separation was carried out with Waters Acquity UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) at a constant temperature of 30 °C. The mobile phase was composed of aqueous solution (0.1% formic acid, A) and acetonitrile (containing 0.1% formic acid, B) and the program of the mobile phase was as follows: 0 min, 40% B; 1 min, 40% B; 1.5 min, 80% B; 2.5 min, 90% B; 4 min, 90% B; 4.1 min, 40% B; 6 min, 40% B, at a flow rate of 0.3 ml/min. The mass spectra were acquired using a triple quadrupole mass spectrometer with ESI ion source in positive mode. The ionization conditions were as follows: drying gas temperature (N2), 300°C; gas flow, 11 L/min (N2); nebulizer pressure, 35 psi (N2). An Agilent Mass Hunter workstation was used for data processing.

The left knee joints were removed on day 13 post STA and fixed in 4% formaldehyde overnight at 4°C. The tissues were decalcified in EDTA and sectioned after embedding in paraffin. 5 μm sections were deparaffinized with xylene, re-hydrated with 100, 95 and 80% ethanol and then stained with hematoxylin and eosin (H&E). Histopathological changes in the knee joints were measured using a semiquantitative scoring system (0–4 scale) to assess synovial hyperplasia and articular inflammatory cells infiltration as previously described (Yamanishi et al., 2002).

150 μL peripheral blood and the hind ankles were collected on day 5 and 13 post KBx/N serum transfer, respectively. For the peripheral blood samples, red blood cells were lysed and cells were prepared as single cell solution. The hind ankles were cut into 1 mm² pieces, and then digested in 10% DMEM containing Collagen IV (1 mg/ml) for 1 h at 37°C. Single cell solution was then prepared after passing through 40 μm cell strainer. 1×10⁶ cells were stained with FITC-conjugated CD11b (BD, United States, 1:500), PE-conjugated F4/80 (BD, United States, 1:400), PerCP-Cy5.5-conjugated Ly6G (BD, United States, 1:400) and APC-conjugated Ly6C (BD, United States, 1:200) to detect macrophage, neutrophil and monocyte. Data were acquired using a BD FACSCalibur flow cytometer (BD Biosciences) and were analyzed by using FlowJo software (Version 7.6.1).

RAW264.7 cells, a mouse macrophage cell line, were cultured in DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin and maintained at 37 °C in 5% CO₂ humidified air. Bone marrow-derived macrophages (BMDMs) generation was performed as following description. In brief, bone marrow cells were harvested from tibias and femurs of C57BL/6 mice and differentiated for 7 days in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin and 50 ng/ml M-CSF. For human peripheral blood mononuclear cells (PBMCs) preparation, 5 ml peripheral venous blood was obtained from healthy donors in West China Hospital. PBMCs were isolated with Human Lymphocyte Separation Medium (LTS1077, Tian Jing, China) according to the manufacturer’s instructions. The experimental protocols were performed according to the approved guidelines established by the Institutional Human Research Subject Protection Committee of the Ethics Committee of West China Hospital, Sichuan University, China.

The effects of EFL2 on RAW264.7 cells, BMDMs and PBMCs viability were evaluated by Cell Counting Kit-8 (CCK8) assay (Dojindo, Japan). Briefly, RAW264.7 cells (1×10⁵ cells/well), BMDMs (1×10⁵ cells/well) and PBMCs (1×10⁶ cells/well) were plated into 96-well plates and then incubated at 37 °C in 5% CO₂ incubator overnight. The medium was disposed next day and cells were incubated with different concentrations of EFL2 (0.1, 0.5, 1, 5, 10, 25, 50 and 100 µM) for 20 h. After disposing the supernatant, 100 μL of culture medium supplemented with 10 μL of CCK8 were added into wells and incubated for an additional 4 h. The optical absorbance at 450 nm was read with a Biotek Eon™ microplate reader (Biotek, Vermont, United States).

RAW264.7 cells (5×10⁵/ml), BMDMs (5×10⁵/ml) and PBMCs (2×10⁶/ml) were plated in 96-well plates and incubated overnight. RAW264.7 cells (5×10⁵/ml) or BMDMs were either solely stimulated by poly:IC (1 μg/ml) (InvivoGen, United States), R837 (20 μg/ml) (Invegen, United States) and the combination of poly:IC and R837, or pretreated with EFL2 (1, 5 and 10 µM) 30 min prior to poly:IC and/or R837 stimulation for 24 h. For the anti-inflammatory molecular mechanism study, RAW264.7 cells (5×10⁵/ml) were plated in 96-well plates and stimulated with R837 (20 μg/ml) with or without the addition of NF-κB inhibitor (JSH-23, 10 μM/ml) for 24 h. After 24 h, the culture supernatant was collected for cytokines measurements.

IL-4, IL-17A and TNF-α in sera from different group mice were measured using CBA Multiplex kit (BD, United States). The concentrations of IL-1β and IL-6 in serum or supernatant were detected by ELISA Kit (MULTI SCIENCES, China) according to the manufacturers’ instructions.

RAW264.7 cells (2×10⁶ cells/well) were plated into 6-well plates and then incubated at 37°C in 5% CO₂ for 24 h. After incubated with various concentrations of EFL2 (1, 5 and 10 µM) for 1 h, cells were then stimulated by R837 (20 μg/ml) for 30 min. Cell lysates were prepared by direct lysis in 20 µL IP buffer (Beyotime Biotechnology, Shanghai, China). After obtaining the whole cell extracts, the cytosolic and nuclear were seperated using Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). BCA Protein Assay Kit (Beyotime Biotechnology, Nantong, China) was applied to determine the protein concentrations. The bands corresponding to p- IKKα/β, IKKα/β, p-IκBα, IκBα, p-p65, p65, p-IRAK4, IRAK4, p-IKKβ, IKKβ, p-IRF5, IRF5, β-actin and Lamin B were visualized using a Western Blot Detection Chemiluminescence Kit (Merck). Protein extracted from the total lysates/cytoplasm and nucleus was quantified in relation to β-actin and Lamin B bands, respectively.

The antibodies used in the experiments were as follows: rabbit anti-mouse IKKα/β polyclonal antibody (1:1,000, Abways, China), rabbit anti-mouse IκBα polyclonal antibody (1:1,000, Abgent, China), rabbit anti-mouse p65 polyclonal antibody (1:1,000, Abgent, China), rabbit anti-mouse phosphorylated IKKα (Ser176)/β (Ser177) (1:1,000, Abways, China), rabbit anti-mouse phosphorylated IκBα (1:1,000, Abcam, United States), rabbit anti-mouse phosphorylated p65 (1:1,000, Abcam, United States), rabbit anti-mouse IRAK4 polyclonal antibody (1:1,000, Proteintech, United States), rabbit anti-mouse phosphorylated IRAK4 (Thr345/Ser346) (1:1,000, Affinity, United States), rabbit anti-mouse IKKβ polyclonal antibody (1:1,000, Abbkine, United States), rabbit anti-mouse phosphorylated IKKβ (Ser177) (1:1,000, Abways, China), rabbit anti-mouse IRF5 polyclonal antibody (1:1,000, Immunoway, United States), rabbit anti-mouse phosphorylated IRF5 (Ser437) (1:1,000, Affinity, United States), anti-mouse actin monoclonal antibody (1:20,000, Transgen, China) and rabbit anti-mouse LaminB monoclonal antibody (1:5,000, Abways, China).

RAW264.7 cells (4×10⁵ cells/well) were seeded into 6-well plates before transfection with the plasmid. When cells confluency reached 60%, RAW264.7 cells were transfected with plasmid IRF5-mouse vector (Origene, United States) using TransEasy™ Transfection Reagent (FOREGENE, China). IRF5 plasmid was suspended with transfection reagent as 1:1.5. Cells were incubated at 37°C for 24 h, then treated with EFL2 and R837 for another 24 h. The supernatant was collected for IL-1β and IL-6 measurements using ELISA kits as described above. For western blot detection, RAW264.7 cells were pretreated with EFL2 (10 µM) for 1 h and stimulated with R837 (20 μg/ml) for 40 min after IRF5 plasmid transfection. Cell lysates were prepared as described above.

P65 and IRF5 translocation activity was analyzed by confocal microscopy. Briefly, 1×10⁵ RAW264.7 cells were plated on coverslips overnight. Cells were pretreated with various concentrations of EFL2 (1, 5 and 10 µM) for 1 h and then stimulated by R837 (20 μg/ml) for 30 min. After washed with ice-cold PBS, RAW264.7 cells were fixed with 4% paraformaldehyde for 20 min at room temperature. The coverslips were washed with PBS with 0.2% tween 20 and blocked with 5% goat serum. Primary antibodies were applied to coverslips overnight at 4°C, followed by secondary antibodies incubation for 1 h at room temperature. After three times wash, coverslips were mounted with Fluoroshield™ with DAPI (Solarbio, China) and images were acquired by confocal microscopy (ZEISS, Germany). Primary antibody: rabbit anti-mouse IRF5 (1:800, Abcam, China); rabbit anti-mouse p65 (1:1,000, Abcam, China). Secondary antibody: Alexa eFluor^®488 conjugated goat anti-rabbit IgG (1:2000, Thermo Fisher Scientific, Carlsbad, United States).

The dissected left ankles were fixed in 4% formaldehyde overnight. Embedding, de-paraffinization, and re-hydration of the ankle sections were the same as described for H&E staining. After treated with 3% hydrogen peroxidase for 10 min, the tissue sections were immersed in a 10 mM sodium citrate buffer (pH 6.0) for 15 min at 95°C, and washed with PBS. The sections were then blocked with 5% goat serum in PBS, and incubated with primary antibodies overnight at 4°C. Primary antibodies were as follows: rabbit anti-mouse IL-1β (1:600, Abcam, China); rabbit anti-mouse IL-6 (1:50, Abcam, China). The signal was visualized using an Envision System (DAKO, United States). The images were then acquired by microscopy (Leica DMil).

Total RNA was isolated from the right ankles using the TRIzol Reagent (Invitrogen) and purified with RNeasy Mini Kit and RNase-Free DNase Set (Qiagen). RNA quality was determined using a spectrophotometer and was reversely transcribed using Wcgene^® mRNA cDNA kit. The complementary DNA was used on the Toll-like Receptor Pathway PCR Array plate (Wcgene^® biotech, China).

Frozen right ankles were pulverized with liquid nitrogen and total RNA was extracted using Trizol reagent (Invitrogen). RNA was reverse transcribed into cDNA using an oligo d(T) primer. The obtained cDNA was applied for quantitative RT-PCR using SYBR Green I-dTTP (Eurogentec). Samples were analyzed in triplicate, and β-actin was used as endogenous controls. Data was analyzed using the 2^−ΔΔCt method and is expressed as “Fold change” (expression relative to housekeeping gene and normalized to reference sample).

Data are presented as the mean ± SEM. Statistical analysis for multiple comparisons was performed by one-way ANOVA followed by Bonferroni’s post-hoc comparisons test using GraphPad Prism 8.0 software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 were considered as significant difference.

Developing anti-arthritic drugs and verifying therapeutic effect can be achieved by taking advantage of animal arthritis models. Although many murine models of arthritis have inflammation effector features, the injection of K/BxN mice serum containing antibodies against glucose-6-phosphate isomerase (G6PI) mainly induce inflammatory arthritis by initiating innate immune system. Unlike collagen-induced arthritis, myeloid cells exert a central role in K/BxN serum-transfer model, but T and B cells are not required (Christensen et al., 2016). Macrophages, neutrophils and Ly6C⁻ nonclassical monocytes are major pivotal cell types which play an essential role in the initiation, progression and resolution of sterile joint inflammation in K/BxN serum-transfer arthritis (STA) model (Solomon et al., 2005; Misharin et al., 2014).

In this study, we evaluate the inhibitory effect of EFL2 on inflammatory arthritis, and also test the therapeutic efficiency of EFL2’s gavage and intraperitoneal (i.p.) injection in STA mice. The clinic scores in STA mice showed a rapidly increased clinic score following with slow remission from day 5–8 (Figure 1B). Meanwhile, histological assessment of the joints demonstrated abundant infiltrated inflammatory cells in the knee of STA mice, accompanied with obvious synovial hyperplasia (Figures 1C–E).

In contrast, DEX administration significantly lowered arthritis clinical scores, and reduced infiltrated cells in the joints of STA mice (Figures 1B–E). In EFL2-treated groups, EFL2 oral administration did not affect the arthritis severity of STA mice at all (Figure 1B). Whereas, i. p. injection with the same dose of EFL2 (40 mg/kg) dramatically improved joint swelling and redness in STA mice (Figure 1B). 15 mg/kg of EFL2, to a moderate extent, alleviated arthritis symptoms, particularly in the acute phase of arthritis (Figure 1B). Mice were sacrificed on day 8 and there were abundant inflammatory cells infiltrating into the joint in STA mice, accompanied with obvious synovial hyperplasia (Figures 1C–E). As shown in Figure 1C, 15 mg/kg of EFL2 only partially reduced the excessive inflammatory cells infiltration and synovium hyperplasia in STA mice. In contrast, high dose of EFL2 (40 mg/kg) or DEX treatment significantly inhibited inflammatory status and inflamed synovium in joints of STA mice (Figures 1C–E).

Inflammatory cytokines (e.g. TNF-α, IL-17, IL-1β and IL-6) are known to be pathogenic factors triggering joint diseases and established synovitis (Martin et al., 2017). The impact of EFL2 on a series of pro-inflammatory cytokines production was also assessed. Interestingly, no significant changes in TNF-α, IL-4 and IL-17A levels were observed among different groups (Figures 1F–H). Unlike TNF-α, IL-1β is absolutely required for disease development in K/BxN serum transfer arthritis (Ji et al., 2002). Meanwhile, IL-6 is also considered as a key inflammatory mediator in the pathogenesis of rheumatoid arthritis (Calabrese and Rose-John, 2014). Over 2-fold levels of IL-1β and IL-6 were detected in the sera of STA mice, which were significantly reduced by EFL2 (40 mg/kg, i. p.) or DEX treatment (Figures 1I,J). In contrast, 15 mg/kg of EFL2 only effectively inhibited IL-6 level of STA mice on day 8 (Figure 1J). Taken together, above results indicate that the intraperitoneal administration of EFL2 is able to ameliorate the disease severity of STA, which might due to the decrease of pro-inflammatory cytokines production in serum.

An UPLC-MS/MS method was successfully developed and applied to a pharmacokinetic study of EFL2 in the plasma after intraperitoneal injection. The main pharmacokinetic parameters of EFL2 were shown in Table 1. After 40 mg/kg intraperitoneal injection, the concentration of EFL2 reached a maximum plasma concentration (C_max) of 4,525.12 ± 1,630.01 ng/ml, and the time to reach the maximum concentration (T_max) was 2.50 ± 1.73 h. The short T max indicated that EFL2 was rapidly absorbed in vivo and produced quick therapeutic effects. The area under the plasma concentration versus time curve from zero to time t (AUC_0–t) was (374.10 ± 163.96) h·ng/ml for EFL2. Besides, EFL2’s half time (t_1/2) value was 21.17 ± 7.11 h, which suggested that EFL2 was slowly eliminated in vivo.

The infiltration of myeloid cells, especially macrophages and neutrophils, is a prominent feature of synovium lesions, and is also positively correlated with the degree of joint erosion (Choy, 2012; Orr et al., 2018). Next, we performed flow cytometry to analyze different myeloid cells in blood and ankle of mice, respectively. Compared with control mice, STA mice showed dramatic increases in the proportions of neutrophil (Ly6G⁺) and inflammatory monocyte (CD11b⁺Ly6C⁺Ly6G⁻) in the circulation (Supplementary Figure S1). In addition, we detected predominant neutrophils infiltration in the ankles of STA mice (Figures 2D,E), accompanied with significantly increased percentages of macrophages and inflammatory monocytes (Figures 2A–C). 30 mg/kg DEX significantly inhibited the increases of these myeloid cells subsets in the ankles of STA mice (Figures 2A–E). While, EFL2, at the dose of 15 mg/kg, remarkably reduced macrophage and inflammatory monocyte influx into the ankles but not neutrophil (Figures 2B,C). 40 mg/kg of EFL2 treatment displayed a more robust suppressive effect on the expansions of these three myeloid cells subsets both in the blood and ankles of STA mice (Supplementary Figure S1, Figures 2A–E).

To assess whereby EFL2 administration prevents the infiltration of these myeloid cells in the ankles, we analyzed the mRNA expressions of known neutrophil chemoattractant, such as CXCL1, CXCL2, CXCL5, and chemokine (C-C motif) ligand CCL3, CCL4 and CCL5 in the ankles of mice. CCL4 and CCL5 mRNA expressions were undetectable in our study. No significantly changes were observed in CXCL2 gene expression among different groups (Figure 2F). In contrast, CXCL1 and CXCL5 mRNA expressions were significantly up-regulated in the ankles of STA mice on day 5, which were remarkably down-regulated by 40 mg/kg EFL2 i. p. treatment (Figure 2F). Meanwhile, this compound also significantly reduced the gene expression of CCL3, known as macrophage trafficking, in STA mice (Figure 2F). In contrast, EFL2, at the dose of 15 mg/kg, only significantly decreased chemoattractant CCL3 mRNA expression (Figure 2F).

Since the infiltration of myeloid cells contributes to increased inflammatory cytokines levels in inflamed joints, we next detected mRNA expression of inflammatory cytokines in the ankles. Compared with control mice, IL-1β and IL-6 mRNA expression increased to 35- and 27-fold in the ankles of STA mice, respectively, which were robustly inhibited by EFL2 i. p. treatment (Figure 2G). Immunohistochemical staining for IL-1β and IL-6 further confirmed EFL2 could inhibit these two cytokines production in the local inflamed joints (Figure 2H). Collectively, these data indicate that EFL2 i. p. administration is able to inhibit inflammatory cells infiltration by suppressing neutrophil and macrophage chemoattractants and suppress their activation to reduce local inflammatory cytokines production.

Toll-Like Receptors (TLRs) are reported to implicate into the initiation, progagation and remission phases of STA (Guo et al., 2018). To identify the most relevant TLR signaling pathway which is implicated into EFL2’s suppressive effect on STA and inflammatory cytokines production, PCR array was performed in this study. As shown in Figure 3A, 44 gene expressions were increased in ankle of STA mice compared with control mice. Recently, emerging newer data indicate the role of TLR7 in the pathogenicity of RA, particular in the aspect of rhematoid synovitis (Kim et al., 2016) and bone erosion (Kim et al., 2019). A recent study showed that TLR7 deficiency alleviated K/BxN serum-induced arthritis by imparing interferon regulatory factor 5 (IRF5) mediated IL-1β and IL-6 produced by macrophages and synovial fibroblasts, respectively (Duffau et al., 2015). We found a 16-fold increase in TLR7 expression at the transcriptional level in STA mice compared with control mice (Figures 3A,B). In addition, mRNA expressions of TLR2 and 9 which mediate an inhibitory role in STA model (Abdollahi-Roodsaz et al., 2013) were also significantly increased in STA mice. Consistently, STA mice displayed up-regulated mRNA expressions of other downstream genes including Myd88, IRAK1/4, Nfkbia/ib, IRF5, Rel, Map3K7 and Mapk8 (Figure 3B). While, 40 mg/kg EFL2 i. p. injection exerted a robust inhibitory effect on above up-regulated genes expression (Figure 3B). The inhibitory effects of TLR2 and TLR9 in STA pathogenesis can not explain the EFL2’s anti-inflammatory role in arthritis. Considering TLR7 mediated-downstream has a promoting role in inflammatory arthritis (Guo et al., 2018), we hypothesized that EFL2 may mainly interfere with TLR7-mediated signaling pathway in STA model to alleviate the inflammatory status in mice.

EFL2 is a latheyran editerpene extracted from Euphorbia lathyris L. seeds with toxicity. We noticed that the peak concontration of EFL2 in sera was equal to 7.03 μM based on Cmax (4.52 ± 1.63 μg/ml) and its molecular weight (C₃₈H₄₂O₉, MW = 642.6). Therefore, we set up the concentrations (from 0.1 to 100 μM) to test the toxicity of EFL2 on these cell lines. As shown in Figure 4. A and D, EFL2, did not display significant cytotoxicity in RAW264.7 cells or primary murine bone marrow-derived macrophages (BMDMs) until its concentration reached 25 μM (Figures 4A,D). In contrast, EFL2 did not affect PBMCs viability even at the high concentration of 100 μM (Figure 4G), suggesting that PBMCs are not sensitive to EFL2 treatment than murine macrophages.

Next, we test EFL2’s anti-inflammatory ability in murine macrophages and human PBMCs in vitro. R837, also known as imiquimod, is a strong TLR7 ligand agonist. It robustly induced IL-1β and IL-6 release in the supernatant of RAW264.7 cells, BMDMs and PBMCs (Figure 4). Notably, EFL2, even at the lower concentration of 0.1 μM, was able to effectively inhibit IL-1β secretion in RAW64.7 or BMDMs (Figures 4B,E). This compound also dose-dependently reduced IL-6 production in murine macrophages (Figures 4C,F). By comparison, the suppressive effect of EFL2 on IL-1β production was prior to that on IL-6 in human PBMCs (Figures 4B,C).

It is shown that TLR3 has a marked synergy role with TLR7 ligand for IL-1β production in BMDMs (Guo et al., 2018). In this study, we applied poly (I:C), a strong TLR3 agonist, to stimulate murine macrophages solely or together with R837. Poly (I:C) itself did not affect IL-1β and IL-6 production in BMDMs, but it significantly increased these two cytokines secretion in RAW264.7 cells. The addition of poly (I:C) did enhance IL-1β and IL-6 production in either R837-stimulated RAW264.7 cells or BMDMs (Figures 4J,K). Various concentrations of EFL2 to different expent decreased the levels of IL-1β and IL-6 in the supernatant of these two murine macrophages co-stimulated by poly (I:C) and R837 (Figures 4J,K). Overall these data demonstrate that EFL2 exerts effective anti-inflammatory role via inhibiting TLR7-induced proinflammatory cytokines production in murine macrophages.

Following TLR7/8 activation, IRAK4 kinase can act with TAK1 to cause IKKβ phosphorylation, subsequently inducing IRF5 phosphorylation and translocation in human monocytes (Cushing et al., 2017). Based on above data, we hypothesize that TLR7-mediated IRF5 signaling pathway is implicated into the molecular mechanisms of EFL2’s anti-inflammatory effect. Similar to the previous findings in human monocyte (Cushing et al., 2017), R837 rapidly triggered the phosphorylation of IRAK4 and IKKβ in RAW264.7 cells (Figures 5A–C), followed with IRF5 phosphorylation at ser437 (Figures 5D,E). Additionally, R837 enhanced IRF5 translocational activity in 1 h, evidenced by the decreased expression in cytoplasm but increased expression in nucleus (Figures 5D,F). Immunofluorescence staining further confirmed that R837 dramatically induced IRF5 translocation from cytoplasm to nucleus (Figure 5G). While, EFL2 pretreatment significantly suppressed the phosphorylation of IRAK4, IKKβ and IRF5 in R837-stimulated RAW264.7 cells (Figures 5A–F). Meanwhile, IRF5 translocation was also effectively blocked by EFL2 at the concentrations of 5 and 10 μM (Figures 5D,F), which was also represented by less IRF5 positive staining in nucleus (Figure 5G).

To further confirm if the blockage of IRF5 activation is an essential process whereby EFL2 decreases pro-inflammatory cytokines reduction, IRF5 plasmid was transfected into RAW264.7 cells. As shown in Supplementary Figure S2, there was a significant upregulation of IRF5 expression in RAW264.7 transfected with IRF5, accompanied with IRF5 expression increase both in cytoplasm and nucleus (Figure 5H). Meanwhile, overexpression of IRF5 significantly reversed the suppressive effect of EFL2 on nuclear translocation of IRF5 (Figures 5H,I). Moreover, EFL2-induced reduction of IL-1β and IL-6 was effectively reversed with overexpression of IRF5 (Figures 5J,K). Overall, above data demonstrate that EFL2 is able to robustly block IRAK4-IKKβ-IRF5 signaling pathway activation, leading to pro-inflammatory cytokines IL-1β and IL-6 reduction.

TLR7 agonist is reported to induce NF-κB activation in myeloid cells (Eng et al., 2018). Considering that IRF5 plasmid transfection did not completely reverse the reduced IL-1β production, we detected the impact of EFL2 on NF-κB activation in R837-stimulated RAW264.7 cells. As shown in Figure 6A, the phosphorylation of IKKα/β and IκB-α rapidly occurs in R837-stimulated RAW264.7 cells (Figures 6A–C), followed by NF-κB subunit p65 phosphorylation and translocation from cytoplasm to nucleus (Figures 6D–G). In TLR7-mediated NF-κB signaling activation, EFL2 effectively inhibited IKKα/β, IκB-α and p65 phosphorylation (Figures 6A,D). In addition, NF-κB subunit p65 translocation activity was also significantly suppressed by this compound (Figures 6D,G), which was further represented by less p65 positive staining in nucleus (Figure 6G). To further confirm if EFL2-induced IL-1β and IL-6 reduction was also NF-κB-dependent in our cell system, we test the effect of JSH-23, a NF-κB inhibitor, on these two cytokines production. Interestingly, JSH-23, to a large extent, blocked R837-induced IL-1β production but only partially reduced IL-6 secretion in RAW 264.7 cells (Figure 6H), suggesting that NF-kB activation was indeed involved into TLR7 activation induced-IL-1β and IL-6 secretion in RAW264.7 cells. Meanwhile, we noticed that EFL2, at the 10 μM, showed a stronger inhibitory ability on IL-1β and IL-6 production compared to JSH-23 (Figure 6H), suggesting NF-kB signaling pathway activation partially contributes to EFL2’s inhibitory effect on inflammatory cytokines release in macrophages.