Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 1. IL-20 accelerated OVX-induced bone loss in orthodontic tooth movement by promoting osteoclastogenesis. (A) Scheme illustrating the establishment of orthodontic tooth movement. (B) The distance of orthodontic tooth movement in the Control group, Force group, and OVX + Force group on day 16. (C) TRAP and Immunohistochemical staining showed that TRAP and IL-20 -positive cells and in the Control group, Force group, and OVX + Force group. Green triangles showed TRAP- positive osteoclasts. Red triangles showed IL-20-positive cells. T: tooth, PL: periodontal ligament, AB: alveolar bone. (D) The quantification of TRAP-positive osteoclasts in the Control group, Force group, and OVX + Force group. (E) Immunohistochemical staining and semiquantification of IL-20 in the Control group, Force group, and OVX + Force group. (F) Double-labelled immunofluorescence staining showed that, in the context of orthodontic force, the expression levels of IL-20 and osteoclast marker protein TRAP increased in the first molar periodontal ligament. (G) The distance of orthodon- tic tooth movement in the OVX group, OVX + Force group, and OVX + Force + risedronate group on day 16. (H) TRAP and immunohistochemical staining showed that TRAP and IL-20 -positive cells and in the OVX group, OVX + Force group, and OVX + Force + risedronate group. (I) The quantification of TRAP-positive osteoclasts in the OVX group, ovariectomy + Force group, and Ovariectomy + Force + risedronate group. (J) Immunohistochemical staining and semiquantification of IL-20 in the OVX group, OVX + Force group, and OVX + Force + risedronate group. (K) Im- munofluorescence staining showed that the expression levels of IL-20 and osteoclast marker protein TRAP increased in the first molar periodontal ligament. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Control, Immunohistochemical staining, Staining, Expressing, Marker

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 2. IL-20 promoted preosteoclast viability via MAPK pathway at the early stage of osteoclast differentiation. Cell viability was examined in M-CSF-induced preosteoclasts by a CCK8 assay. (A,B) The expression of IL-20 in M-CSF-induced preosteoclasts was determined by cellular im- munofluorescence staining on day 2. (C–F) The mRNA expression levels of IL-20, IL-20RA, IL-20RB, and IL-22RA1 in preosteoclasts were evaluated by qRT-PCR after 2 days of M-CSF treatment. (G) Cell viability detection in preosteoclasts treated with a gradient of concentrations of IL-20 on days 1, 3, 5, and 7. (H) Preosteoclasts were treated with 2 ng/mL IL-20 or anti-IL-20 antibody. The levels of phosphorylation of proteins in the IL-20-mediated signaling pathway, including p38, phos-pho-p38, ERK, phospho-ERK, JNK, and phospho-JNK, were analyzed using Western blotting. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. * p < 0.05 vs. the 0 ng/mL IL-20 group. ns p > 0.05 vs. the 0 ng/mL IL-20 group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: CCK-8 Assay, Expressing, Staining, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 3. IL-20 had no effect on osteoclast formation at the early stage of osteoclast differentiation. (A) Scheme illustrating bone marrow-derived macrophages were cultured in osteoclast medium containing M-CSF and IL-20 or anti-IL-20 antibody at the early stage of osteoclast differentiation, and then induced with the presence of 10, 30, or 60 ng/mL RANKL. The control group included M-CSF-induced preosteoclasts induced with 10, 30, or 60 ng/mL RANKL. (B) TRAP staining was performed, and the number and size of TRAP-positive osteoclasts with more than three nuclei were quantified on day 6. IL-20-block group meant that cells were treated with IL-20 or anti-IL-20 antibody. (C) A bone resorption pit assay was performed to detect osteoclast function, and bone resorption pits were counted, and the area and number of bone resorption were quantified on day 6. IL-20- block group meant that cells were treated with IL-20 and anti-IL-20 antibody. (D) M-CSF-induced preosteoclasts were cultured in osteoclast medium containing different concentrations of IL-20 or anti-IL-20 antibody without RANKL. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Derivative Assay, Cell Culture, Control, Staining, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 4. IL-20 promoted RANKL-induced osteoclast differentiation and bone resorption function. (A) Scheme illustrating M-CSF-induced preosteoclasts were cultured in osteoclast medium containing IL-20 or anti-IL-20 antibody at the late stage of osteoclast differentiation in the presence of 10, 30, or 60 ng/mL RANKL. (B) TRAP staining was performed, and the number and size of TRAP-positive osteoclasts with more than three nuclei were quantified at the late stage of differentiation on day 6. The control group included M-CSF-induced preosteoclasts induced with 10, 30, or 60 ng/mL RANKL. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. (C) A resorption pit assay was performed to detect osteoclast function, and resorption pits were counted at the late stage of differentiation on day 6, and the area and number of bone resorption were quantified at the stage of differentiation. IL-20-block group meant that cells were treated with IL-20 and anti-IL-20 antibody. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Cell Culture, Staining, Control, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 5. IL-20 promoted the expression of osteoclast-specific genes and proteins via NF-κB pathway during RANKL-induced osteoclast differentiation. Preosteoclasts were stimulated with IL-20 or NF-κB pathway inhibitor TPCA-1. (A,B) IL-20 promoted phospho-P65 nuclear translocation and was blocked by TPCA-1 in preosteoclasts after 1 h treatment, with or without RANKL. (C) The levels of phosphorylation for proteins in the NF-κB pathway, including the IKKα/β, IκB-α, and P65 proteins in preosteoclasts were detected using Western blotting. (D) The levels of activated proteins in preosteoclasts, including the RANK, TRAF6, c-Fos, and NFATc1 proteins without RANKL were detected using Western blotting. (E) The protein expression levels of TRAP, Cathepsin K, RANK, TRAF6, c-Fos and NFATc1 in osteoclasts were examined using Western blotting after 6 days of IL-20 treatment. (F–K) The mRNA expression levels of TRAP, Cathepsin K, MMP9, MT1-MMP, c-Fos, and NFATc1 in osteoclasts were detected by qRT-PCR after 6 days of IL-20 treatment. * p < 0.05 vs. the 0 ng/mL IL-20 group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Expressing, Translocation Assay, Phospho-proteomics, Western Blot, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Dual Role of Interleukin-20 in Different Stages of Osteoclast Differentiation and Its Osteoimmune Regulation during Alveolar Bone Remodeling.

doi: 10.3390/ijms24043810

Figure Lengend Snippet: Figure 6. Therapeutic effect of IL-20 on orthodontic tooth movement. (A,B) Micro-CT showed the distance of orthodontic tooth movement between first and second molars treated with local infusion of IL-20 or an-ti-IL-20 antibody, seven days after the application of orthodontic force. OTM + IL- 20-block group meant that rats were locally infused with anti-IL-20 antibody. (C,D) TRAP staining and the quantification of TRAP-positive osteoclasts in the OTM group, OTM + IL-20 group, and OTM + anti-IL-20 antibody group. Green triangles showed TRAP-positive osteoclasts. OTM + IL-20- block group meant that rats were locally infused with anti-IL-20 antibody. (E,F) Immunofluorescence staining showed that the expression levels of osteoclast marker protein RANK in the first molar periodontal ligament after the application of orthodontic force. OTM + IL-20-block group meant that rats were locally infused with anti-IL-20 antibody. (G,H) Immunofluorescence staining showed that the expression levels of MCP-1 and CD11b in the first molar periodontal ligament after the application of orthodontic force. * p < 0.05 vs. the control group. n = 6.

Article Snippet: After the treatment of IL-20, anti-IL-20 antibody, or TPCA-1 (MCE, Monmouth Junction, NJ, USA), we obtained protein from the samples with cold RIPA containing 1% protease inhibitor cocktail and 1% phosphatase inhibitors.

Techniques: Micro-CT, Blocking Assay, Staining, Expressing, Marker, Control

Journal: JCI Insight

Article Title: IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

doi: 10.1172/jci.insight.181906

Figure Lengend Snippet: ( A ) Splenic images, ( B ) spleen weights, ( C ) serum levels of anti-dsDNA antibodies, ( D ) H&E staining of kidney, and ( E ) renal IgG and IgM deposition in WT or IL-17B –KO mice treated with IMQ. ( F ) Splenic images, ( G ) spleen weights, ( H ) serum levels of anti-dsDNA antibodies, ( I ) H&E staining of kidney, and ( J ) renal IgG and IgM deposition in IMQ-induced WT mice treated with vehicle or rmIL-17B. Scale bars: 100 μm. The data are shown as the mean ± SEM and are representative of 3 independent experiments ( n = 6 mice/group). * P < 0.05, ** P < 0.01 by 2-tailed Student’s t test.

Article Snippet: To explore the effect of rmIL-17B on the pathogenesis of MRL/ lpr mice, female MRL/ lpr mice (10 weeks old) were randomly divided into 2 groups: one group ( n = 6) was injected with rmIL-17B, the other group ( n = 6) was injected with vehicle. rmIL-17B (0.5 μg/g; MCE) or vehicle was injected into mice via the tail vein (twice per week).

Techniques: Staining

Journal: JCI Insight

Article Title: IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

doi: 10.1172/jci.insight.181906

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection plot of spleen cells from IMQ-treated WT ( n = 2) and IL-17B –KO mice ( n = 2). ( B ) Proportion of cell types in the splenocytes of IMQ-treated WT ( n = 2) or IL-17B –KO mice ( n = 2). ( C ) KEGG analysis of splenic GC B cells from IMQ-treated WT and IL-17B –KO mice. Representative flow cytometry images and statistical analysis of the percentages of ( D ) splenic GC B cells (B220 + GL-7 + CD95 + ), ( E ) plasma cells (CD138 + B220 – ), and the expression of ( F ) CD86 and ( G ) CD69 on B220 + B cells in IMQ-treated WT or IL-17B –KO mice. The percentages of ( H ) spleen GC B cells, ( I ) plasma cells, and the expression of ( J ) CD86 and ( K ) CD69 on B220 + B cells in IMQ-induced WT mice treated with vehicle or rmIL-17B. The data are shown as the mean ± SEM and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 by 2-tailed Student’s t test. NS, P > 0.05.

Article Snippet: To explore the effect of rmIL-17B on the pathogenesis of MRL/ lpr mice, female MRL/ lpr mice (10 weeks old) were randomly divided into 2 groups: one group ( n = 6) was injected with rmIL-17B, the other group ( n = 6) was injected with vehicle. rmIL-17B (0.5 μg/g; MCE) or vehicle was injected into mice via the tail vein (twice per week).

Techniques: Flow Cytometry, Clinical Proteomics, Expressing

Journal: JCI Insight

Article Title: IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

doi: 10.1172/jci.insight.181906

Figure Lengend Snippet: WT mice spleen B cells were pretreated with rmIL-17B for 12 hours and stimulated with LPS (100 ng/mL), R848 (1 μg/mL), and CpG-1826 (1 μM). ( A ) CD40, ( B ) CD69, and ( C ) CD86 expression at 24 hours. ( D ) IL-12 and ( E ) TNF-α mRNA levels at 6 hours. ( F ) Total (t-) and phosphorylated (p-) p65, p38, JNK, and Erk at 30 or 60 minutes after R848 stimulation. ( G ) CD40, ( H ) CD69, and ( I ) CD86 expression at 24 hours, and ( J ) MX1, ( K ) OAS1, and ( L ) IFIT1 mRNA levels at 6 hours in WT mice spleen B cells pretreated with rmIL-17B for 12 hours and stimulated with IFN-α (1000 U/mL). The data are shown as the mean ± SEM and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test ( A – E and G – I ) or 2-way ANOVA with Bonferroni’s mutiple-comparison test ( J – L ). NS, P > 0.05.

Article Snippet: To explore the effect of rmIL-17B on the pathogenesis of MRL/ lpr mice, female MRL/ lpr mice (10 weeks old) were randomly divided into 2 groups: one group ( n = 6) was injected with rmIL-17B, the other group ( n = 6) was injected with vehicle. rmIL-17B (0.5 μg/g; MCE) or vehicle was injected into mice via the tail vein (twice per week).

Techniques: Expressing, Comparison

Journal: JCI Insight

Article Title: IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

doi: 10.1172/jci.insight.181906

Figure Lengend Snippet: ( A ) PCA plot, ( B ) volcano plot, and ( C ) KEGG analysis of metabolites generated from serum lipid profiles of IMQ-treated WT ( n = 6) and IL-17B –KO mice ( n = 6). ( D ) The FASN intensity on each cell from IMQ-treated WT mice ( n = 2) and IL-17B –KO mice ( n = 2) was expressed as z score–normalized expression. Dashed-line circles indicate GC B cells. ( E and F ) Western blot and flow cytometry analysis of FASN expression in B cells after different time periods of rmIL-17B treatment. ( G ) Quantitative PCR analysis was performed to assess the expression of FASN in B cells following treatment with varying concentrations of rmIL-17B. ( H – M ) B cells, isolated from the spleen of WT mice or FASN +/– mice, were stimulated with LPS, R848, and CpG-1826. Expression of ( H ) CD40, ( I ) CD69, and ( J ) CD86 at 24 hours. mRNA levels of ( K ) IL-12 and ( L ) TNF-α at 6 hours. ( M ) Phosphorylation of p65, p38, JNK, and Erk. ( N – S ) B cells, isolated from the spleen of WT mice or FASN +/– mice, were stimulated with IFN-α. ( N ) CD40, ( O ) CD69, and ( P ) CD86 expression at 24 hours. ( Q ) MX1, ( R ) OAS1, and ( S ) IFIT1 mRNA levels at 6 hours. ( T – V ) B cells, isolated from the spleen of WT mice or FASN +/– mice, were stimulated with R848, and the ( T and U ) OCR was detected by seahorse, and the ( V ) ATP concentration was detected by ELISA. Murine splenic B cells were pretreated with rmIL-17B for 12 hours and stimulated with R848, and ( W and X ) OCR was detected. The data are shown as the mean ± SEM and are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test ( F and G ) or 2-tailed Student’s t test ( H – L , N – S , U , V , and X ). NS, P > 0.05.

Article Snippet: To explore the effect of rmIL-17B on the pathogenesis of MRL/ lpr mice, female MRL/ lpr mice (10 weeks old) were randomly divided into 2 groups: one group ( n = 6) was injected with rmIL-17B, the other group ( n = 6) was injected with vehicle. rmIL-17B (0.5 μg/g; MCE) or vehicle was injected into mice via the tail vein (twice per week).

Techniques: Generated, Expressing, Western Blot, Flow Cytometry, Real-time Polymerase Chain Reaction, Isolation, Phospho-proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison