Fig 1: CRP upregulated HIF1α to interfered Leflunomide-AHR-CRP signaling in vitro. a Levels of CRP and HIF1α in hepatocytes from non-immunized (NI) rats, CRPH and CRPL CIA rats. n = 9 for each group. b Level of HIF1α in human normal hepatocytes (THLE-2 cells) transfected with vehicle, CRP siRNA and negative control siRNA (NC siRNA), respectively. c Level of ARNT binding with HIF1α in THLE-2 cells after the transfection. d Level of HIF1α in THLE-2 cells transfected with vehicle, empty vector and CRP overexpressing vector, respectively. THLE-2 cells incubated in hypoxia were used as a positive control. e Level of ARNT binding with HIF1α in THLE-2 cells after the transfection. f Level of ARNT binding with HIF1α or AHR in THLE-2 cells transfected with CRP siRNA in the presence of Leflunomide (LEF). g Level of ARNT binding with HIF1α or AHR in THLE-2 cells transfected with CRP overexpressing vector in the presence of Leflunomide (LEF). h Proposed mechanism underlying the dysfunction of Leflunomide-AHR-CRP signaling in CRPH RA. Briefly, high level of CRP could trigger upregulation of HIF1α, which competes with AHR for ARNT association, leading to the dysfunction of Leflunomide-AHR-CRP signaling in CRPH RA. i Level of ARNT binding with HIF1α in IL-6-pretreated THLE-2 cells transfected with HIF1α siRNA and NC siRNA, respectively. Pretreatment of IL-6 induced the overexpression of CRP in THLE-2 cells. j Level of ARNT binding with AHR in IL-6-pretreated THLE-2 cells transfected with HIF1α siRNA in the presence of Leflunomide (LEF). k Relative CRP mRNA (left) and CRP release (right) in IL-6-pretreated THLE-2 cells after the treatment. *P < 0.05 as determined by one-way ANOVA with a post hoc test. Each experiment was repeated three times. Source data are provided as a Source Data file
Fig 2: Effects of Leflunomide on AHR genomic signaling and CRP expression in vitro. a Pull-down assay for interaction between Flag-tagged AHR (Flag-AHR) and Leflunomide (LEF, left) or A77 1726 (right). Flag-AHR was incubated with Leflunomide, A77 1726 and vehicle (DMSO), respectively. Small molecules captured by Flag-AHR were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). b Pull-down assay for interaction between AHR mutants and Leflunomide (LEF). Flag-AHR and AHR mutants (Flag-H291A, Flag-K303A, Flag-V381A and Flag-H291A/K303A) was incubated with Leflunomide or vehicle (DMSO). c Binding of ARNT with AHR determined by immunoprecipitation in human normal hepatocytes (THLE-2) incubated with vehicle (DMSO), Leflunomide (LEF) and A77 1726 at a series of concentrations, respectively. d Relative levels of CRP mRNA (left) and CRP release (right) in THLE-2 cells incubated with Leflunomide (LEF), A77 1726 and vehicle (Veh, DMSO), respectively. e Binding of ARNT with AHR in IL-6-pretreated THLE-2 cells with incubation of Leflunomide (LEF) and vehicle (DMSO) in the presence of CRP siRNA or negative control siRNA (NC siRNA), respectively. Pretreatment of IL-6 induced the overexpression of CRP in THLE-2 cells. f Relative levels of CRP mRNA (left) and CRP release (right) in IL-6-pretreated THLE-2 cells incubated with vehicle (DMSO), Leflunomide (LEF) and A77 1726, respectively. *P < 0.05 as determined by one-way ANOVA with a post hoc test. NS: no significance. Each experiment was repeated three times. g Besides the well-known immunomodulatory action via A77 1726, Leflunomide itself could activate AHR genomic signaling to inhibit CRP production and then attenuate bone erosion in CRPL RA (left), whereas the Leflunomide-AHR-CRP signaling was dysfunctional in CRPH RA (right). Source data are provided as a Source Data file
Fig 3: Differential responsiveness to Leflunomide among CIA rats. a The representative three-dimensional microCT images and clinical arthritic scores of the hind paws from PBE+ (n = 73) and PBE− (n = 77) CIA rats before (baseline, BL) and after Leflunomide treatment. Non-immunized (NI) rats (n = 20) were used as controls of the CIA rats. Scale bar, 5.0 mm. *P < 0.05 as determined by repeated-measures ANOVA with a post hoc test. b Relative levels of DHODH activity and proliferation of T and B lymphocytes in synovial fluid normalized to the corresponding baseline in PBE+ (n = 73) and PBE− (n = 77) CIA rats. *P < 0.05 as determined by one-way ANOVA with a post hoc test, NS: no significance. c The serum baseline CRP and TRAP5b levels in PBE− (n = 77) and PBE+ (n = 73) rats. CRPL: relatively lower CRP level (CRPLower, 52.45 ± 7.54 mg L−1). CRPH: higher CRP level (CRPHigher,91.24 ± 10.12 mg L−1). *P < 0.05 as determined by two-sided t-test. d Relative changes of serum CRP (left) and TRAP5b (right) from the corresponding baseline in CRPH rats (n = 73) and CRPL (n = 77) rats. *P < 0.05 for day 14 or day 28 versus BL in CRPL and CRPH, #P < 0.05 for CRPH versus CRPL at day 14 and day 28, as determined by repeated-measures ANOVA with a post hoc test. e Bone resorption parameters including osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (Oc.N/BS) in CRPH (n = 73) and CRPL (n = 77) rats after normalization to the corresponding baseline. *P < 0.05 as determined by one-way ANOVA with a post-hoc test. Source data are provided as a Source Data file
Fig 4: ACF facilitated Leflunomide attenuating bone erosion in CRPH CIA rats. a The diagram of the experimental design. Briefly, SD rats were immunized with bovine type II collagen for 21 days to establish the CIA model. The CIA rats with CRP level within the established reference range for CRPH rats (91.24 ± 10.12 mg L−1) were administered with vehicle (Veh), Leflunomide (LEF, 10.0 mg kg−1 per day), ACF (1.0 mg kg−1 per day), and the combination of Leflunomide and ACF (LEF + ACF, LEF, 10,0 mg kg−1 per day; ACF, 1.0 mg kg−1 per day) for 28 days, respectively. b The representative reconstructed three-dimensional micro-CT images and clinical arthritic score of the hind paws from CRPH rats before (baseline, BL) and after the treatment. Non-immunized (NI) rats were used as the controls of the CIA rats. Scale bar, 5.0 mm. *P < 0.05 as determined by repeated - measures ANOVA with a post hoc test. c Levels of serum CRP and TRAP5b in CRPH rats before (baseline, BL) and after the treatments. d Bone resorption parameters including osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (Oc.N/BS) in CRPH rats before (baseline, BL) and after the treatments. e Bone mass parameters including bone mineral density (BMD) and bone volume per total volume (BV/TV) in CRPH rats before (baseline, BL) and after the treatments. *P < 0.05 as determined by one-way ANOVA with a post hoc test. n = 9 for each group. Source data are provided as a Source Data file
Fig 5: ACF re-activated Leflunomide-AHR-CRP signaling in vitro. a Binding of ARNT with HIF1α and level of VEGF in IL-6-pretreated THLE-2 cells incubated with vehicle (DMSO) or ACF (5.0 μM). Pretreatment of IL-6 induced the overexpression of CRP in THLE-2 cells. THLE-2 cells incubated in hypoxia were used as a positive control. b Binding of ARNT with AHR in IL-6-pretreated THLE-2 cells incubated with vehicle (DMSO), Leflunomide (LEF, 10 μM), ACF (5.0 μM) and the combination of Leflunomide and ACF (LEF + ACF, LEF, 10 μM; ACF, 5.0 μM), respectively. c Relative CRP mRNA and CRP release in IL-6-pretreated THLE-2 cells after the treatment. d The diagram of the experimental design. Briefly, THLE-2 cells were pretreated with IL-6 and then incubated with vehicle (DMSO), Leflunomide (LEF, 10 μM), ACF (5.0 μM) and the combination of Leflunomide and ACF (LEF + ACF, LEF, 10 μM; ACF, 5.0 μM), respectively. After 8 h, the medium was replaced, and the THLE-2 cells were continuously cultured for another 48 h. The conditioned mediums were collected for culturing monocytes in the presence of macrophage colony stimulating factor (M-CSF). e TRAP staining (upper) and pit formation assay (bottom) in monocytes incubated with the conditioned mediums. Scale bar, 100 μm. f Quantitative determination of TRAP-positive (TRAP+) multinuclear cells per well and percentage of pit area from pit formation assay. g Relative TRAP5b mRNA level in monocytes incubated with the conditioned mediums. *P < 0.05 as determined by one-way ANOVA with a post hoc test. Each experiment was repeated three times. Source data are provided as a Source Data file
Supplier Page from Abcam for Anti-C Reactive Protein antibody