Journal: Journal of Leukocyte Biology

Article Title: IL‐38 prevents induction of trained immunity by inhibition of mTOR signaling

doi: 10.1002/JLB.3A0220-143RRR

Figure Lengend Snippet: Effect of IL‐38 on the inflammatory response to LPS . ( A ) Experimental outline. Mice received on three consecutive days 1 μg IL‐38 i.p. or vehicle. All mice received on day 5 mg/kg LPS i.p. and were sacrificed 4 h later. ( B ) Abdominal wall temperature of mice at sacrifice. ( C ) Cytokine levels in plasma and blood lysate at sacrifice. ( D ) Spontaneous TNFα and IL‐6 production in supernatants from whole blood, bone marrow, and splenocytes after 24 h ex vivo culture. N = 5, mean ± sem , cytokine production normalized to monocyte numbers * P < 0.05, ** P < 0.01, ns not significant

Article Snippet: Mice received 1 μg of bioactive , human recombinant IL‐38 (3‐152) (Biotechne, Minneapolis, MN, USA) i.p. in sterile saline in a 200 μl volume or only saline for three consecutive days.

Techniques: Ex Vivo

Journal: Journal of Leukocyte Biology

Article Title: IL‐38 prevents induction of trained immunity by inhibition of mTOR signaling

doi: 10.1002/JLB.3A0220-143RRR

Figure Lengend Snippet: Effect of IL‐38 on the induction of trained immunity by β‐glucan . ( A ) The experimental outline is comparable to Figure , but now after the first dose of IL‐38, 1 mg of β‐glucan (or vehicle) was administered i.p. ( B ) Temperature at sacrifice. ( C ) Cytokine levels in plasma and blood lysate at sacrifice. ( D ) Spontaneous TNFα and IL‐6 production in supernatants from whole blood, bone marrow, and splenocytes after 24 h ex vivo culture. N = 5, mean ± sem , cytokine production normalized to monocyte numbers * P < 0.05, ** P < 0.01, *** P < 0.001, ns not significant

Article Snippet: Mice received 1 μg of bioactive , human recombinant IL‐38 (3‐152) (Biotechne, Minneapolis, MN, USA) i.p. in sterile saline in a 200 μl volume or only saline for three consecutive days.

Techniques: Ex Vivo

Journal: Journal of Leukocyte Biology

Article Title: IL‐38 prevents induction of trained immunity by inhibition of mTOR signaling

doi: 10.1002/JLB.3A0220-143RRR

Figure Lengend Snippet: Epigenetic effect of IL‐38 on the induction of trained immunity by β‐glucan . ( A ) The experimental outline is comparable to Figure , but now no LPS administration (except for panel C). ( B ) TNFα and IL‐1β production in supernatants from whole blood, bone marrow, and splenocytes after 24 h culture with 100 ng/ml LPS and 1 h 10 μM Nigericin. ( C ) RNA expression in bone marrow after 4 h in vivo LPS. ( D ) Percentage of H3K4me3 at the promoters of genes displayed. N = 12, mean ± sem , cytokine production normalized to monocyte numbers, * P < 0.05, ** P < 0.01, ns not significant

Article Snippet: Mice received 1 μg of bioactive , human recombinant IL‐38 (3‐152) (Biotechne, Minneapolis, MN, USA) i.p. in sterile saline in a 200 μl volume or only saline for three consecutive days.

Techniques: RNA Expression, In Vivo

Journal: Journal of Leukocyte Biology

Article Title: IL‐38 prevents induction of trained immunity by inhibition of mTOR signaling

doi: 10.1002/JLB.3A0220-143RRR

Figure Lengend Snippet: Effect of IL‐38 on β‐glucan‐dependent phosphorylation of the mTOR pathway . ( A ) After naïve bone marrow incubation with IL‐38 (100 ng/ml) or vehicle for 30 min and subsequent β‐glucan (5 μg/ml) or vehicle for 4 h Western blot for indicated proteins was performed on the cell lysate. Actin and total proteins were used as loading control. The graphs represent the phosphorylated protein divided by the total protein in arbitrary units, N = 3 ( B ) Mice were treated following the protocol of Figure . Four hours after LPS stimulation serum lactate levels were determined, and ( C ) bone marrow was isolated and plated for 72 h after which lactate levels were determined in the supernatant. N = 5 (B‐C), mean ± sem , * P < 0.05, ** P < 0.01, **** P < 0.0001, ns not significant

Article Snippet: Mice received 1 μg of bioactive , human recombinant IL‐38 (3‐152) (Biotechne, Minneapolis, MN, USA) i.p. in sterile saline in a 200 μl volume or only saline for three consecutive days.

Techniques: Incubation, Western Blot, Isolation

Journal: Journal of Leukocyte Biology

Article Title: IL‐38 prevents induction of trained immunity by inhibition of mTOR signaling

doi: 10.1002/JLB.3A0220-143RRR

Figure Lengend Snippet: Effect of IL‐38 ( IL‐1F10 ) single nucleotide polymorphism (SNP) rs58965312 on β‐glucan training response and plasma IL‐38 . ( A ) Monocytes were trained with β‐glucan or control for 24 h. One week later, cells were stimulated for 24 h with 10 ng/ml LPS and IL‐6 and TNFα were determined in supernatant. Induction of cytokine production (β‐glucan/control) was determined. ( B ) Plasma levels of IL‐38 in corresponding individuals. Wt = CC = 35, He = CT = 53, Ho = TT = 30, mean ± sem , * P < 0.05, ns not significant

Article Snippet: Mice received 1 μg of bioactive , human recombinant IL‐38 (3‐152) (Biotechne, Minneapolis, MN, USA) i.p. in sterile saline in a 200 μl volume or only saline for three consecutive days.

Techniques:

Journal: Biochemistry and Biophysics Reports

Article Title: IL-38: A new factor in rheumatoid arthritis

doi: 10.1016/j.bbrep.2015.10.015

Figure Lengend Snippet: Characteristics of human IL-38. (A) Western blotting analysis using anti-human IL-38 mAb (H127C) showed that human IL-38 cDNA-transfected 293T cells expressed approximately 18 kDa IL-38 protein in the cells and supernatants. Line 1: Cell lysate of vector-transfected 293T cells. Line 2: Cell lysate of human IL-38 cDNA-transfected-transfected 293T cells. Line 3: Supernatant of vector-transfected 293T cells. Line 4: Supernatant of human IL-38 cDNA-transfected-transfected 293T cells. As control, recombinant human IL-38 (rhIL-38) protein with His tag at C-terminal was used. (B) Standard curve of human IL-38 sandwich ELISA using anti-human IL-38 mAbs (H127C and H160A). (C) Caspase-1, chymase, and PR3 can cleave recombinant human pro-IL-1β (hpro-IL-1β), but not rhIL-38 protein. Western blotting analysis was performed using rabbit anti-human pro-IL-1β polyclonal Ab (Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-human IL-38 polyclonal Ab (established at our laboratory).

Article Snippet: Digestion of recombinant human IL-38 and pro-IL-1β protein (Sino Biological Inc., Beijing, China) by recombinant caspase 1, chymase, and PR3 (Sigma, St. Louis, MO, USA) was performed as reported previously , .

Techniques: Western Blot, Transfection, Plasmid Preparation, Recombinant, Sandwich ELISA

Journal: Biochemistry and Biophysics Reports

Article Title: IL-38: A new factor in rheumatoid arthritis

doi: 10.1016/j.bbrep.2015.10.015

Figure Lengend Snippet: IL-38 expression in RA serum and synovium. (A) Serum levels of IL-38 in RA, OA patients and healthy donors. Serum levels of IL-38 was examined by IL-38 ELISA. Twenty-one of 137 RA (15.3%), one of 26 OA patients (3.9%) and 5 of 56 controls (8.9%) were elevated above the detection limit. Detection limit is 9.35 ng/ml. (B) IL-38 is expressed in RA, but not OA synovium. Synovial tissues were obtained from 7 RA patients and 2 OA patients. Synovial tissues were immunostained with anti-human IL-38 mAb [H127C, mouse IgG2b] (b, c, d) or isotype-matched control mouse IgG2b Ab (a). Representative sections of synovial tissues from 2 RA patients (b, c) and a patient with OA (d) are shown. Original magnification: ×200.

Article Snippet: Digestion of recombinant human IL-38 and pro-IL-1β protein (Sino Biological Inc., Beijing, China) by recombinant caspase 1, chymase, and PR3 (Sigma, St. Louis, MO, USA) was performed as reported previously , .

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biochemistry and Biophysics Reports

Article Title: IL-38: A new factor in rheumatoid arthritis

doi: 10.1016/j.bbrep.2015.10.015

Figure Lengend Snippet: IL-38 gene deficiency enhances joint inflammation in RA mouse model. Arthritis was initiated in female IL-38 −/− mice and control B6 WT mice via intraperitoneal administration of K/BxN mouse serum. (A) IL-38 mRNA in ankle joints (8 ankles/group from two separate experiments) was determined before or 8 days after K/BxN serum transfer. * p <0.05, normal vs. inflamed joints. (B) Clinical score of K/BxN serum transfer arthritis in WT and IL-38 −/− mice on a 0–16 scale ( n =5/group), * p <0.05, ** p <0.01, WT vs. IL-38 −/− mice. Data are representative of at least 2 separate experiments. (C) Histomorphometric quantification of arthritic tissue. Data were pooled in two independent experiments (10 ankles/group from two separate experiments). * p <0.05, WT vs. IL-38 −/− mice. (D) Histopathologic findings in the ankle joints of representative WT (left) and IL-38 −/− mice (right). Hematoxylin and eosin stained; original magnification ×40. (E) Cytokine mRNA in the ankle joints (10 ankles/group from two separate experiments) was examined at day 7 or 8 arthritis. * p <0.05, WT vs. IL-38 −/− mice. Values in A–C and E are the mean±SEM.

Article Snippet: Digestion of recombinant human IL-38 and pro-IL-1β protein (Sino Biological Inc., Beijing, China) by recombinant caspase 1, chymase, and PR3 (Sigma, St. Louis, MO, USA) was performed as reported previously , .

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Expression of IL‐38 and its receptor IL‐36R in murine CD4 + CD25 + Tregs after LPS stimulation. CD4 + CD25 + Tregs were isolated from murine splenocytes. (A, B) IL‐38 mRNA levels in CD4 + CD25+Tregs stimulated with 0.5, 1 or 5 μg/mL LPS for 12, 24 or 48 h. C, IL‐36R mRNA expression in CD4 + CD25+Tregs stimulated with LPS (5 μg/mL) for 48 h. (D, E) IL‐38 protein levels in CD4 + CD25 + Tregs stimulated with 0.5, 1 or 5 μg/mL LPS for 12, 24 or 48 h. F, Mean fluorescence intensity (MFI) of IL‐38 expression in CD4 + CD25 + Tregs stimulated with LPS (5 μg/mL) for 48 h, as analysed using laser scanning confocal microscopy. * P < .05, ** P < .01, vs the PBS control group (n = 3 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Expressing, Isolation, Fluorescence, Confocal Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Effects of IL‐38 on Foxp3 and CD152 (CTLA‐4) expression in murine CD4 + CD25 + Tregs. CD4 + CD25+Tregs were isolated from murine splenocytes. (A, E) Expression of Foxp3 and CD152 in murine CD4 + CD25+Tregs treated with PBS or 50, 100 or 200 ng/mL IL‐38 for 48 h. * P < .05, ** P < .01, *** P < .001 vs the PBS control group. (B, E) Expression of Foxp3 and CD152 in murine CD4 + CD25 + Tregs stimulated with 100 ng/mL IL‐38 for 12, 24 or 48 h. * P < .05, ** P < .01, vs the PBS control group. (C, F) Foxp3 and CD152 expression in CD4 + CD25 + Tregs incubated for 48 h with LPS (5 μg/mL) and IL‐38 (50, 100, or 200 ng/mL). ** P < .01 vs the PBS control group; ## P < .01, ### P < .001 vs. the LPS group. (D, G) Expression of Foxp3 and CD152 in CD4 + CD25+Tregs isolated from the spleens of mice treated intraperitoneally with IL‐38 (1 μg per mouse) or PBS (the control group), which underwent the caecal ligation or puncture (CLP) procedure 2 h later. Mice in the sham group were subjected to the same procedure without CLP. * P < .05, ** P < .01 vs the negative control (NC) group; # P < .05, ## P < .01 vs the CLP group (n = 6 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Expressing, Isolation, Incubation, Ligation, Negative Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Influence of IL‐38 on the production of IL‐10 and TGF‐β1 in murine CD4 + CD25 + Tregs. CD4 + CD25 + Tregs were isolated from murine splenocytes. (A‐D) IL‐10 and TGF‐β1 levels in supernatants from murine CD4 + CD25 + Tregs treated with IL‐38 at 50, 100 or 200 ng/mL for 12, 24 or 48 h, as measured by ELISA. * P < .05, *** P < .01, *** P < .001 vs the PBS control group. (E, F) IL‐10 and TGF‐β1 levels in supernatants from CD4 + CD25 + Tregs stimulated for 48 h with LPS (5 μg/mL) together with IL‐38 at 50, 100 or 200 ng/mL, as assessed by ELISA. * P < .05 vs the PBS control group; ## P < .01 vs the LPS group. (G, H) Levels of IL‐10 and TGF‐β1 in mice intraperitoneally injected withIL‐38 (1 μg per mouse) or PBS (the control group), which underwent CLP 2 h later. * P < .05, ** P < .01 vs the NC group; ## P < .01 vs the CLP group (n = 6 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Effect of IL‐38 on the suppressive activity of murine CD4 + CD25 + Tregs isolated from murine splenocytes. CD4 + CD25 − T cells and CD4 + CD25 + Tregs were isolated from murine splenocytes. (A, B, D, E) Proliferation of CD4 + CD25 − T cells examined by CFSE staining after stimulation for 48 h with IL‐38 at 50, 100 or 200 ng/mL in the presence or absence of LPS (5 μg/mL). CD4 + CD25 + Tregs were incubated with CFSE‐labelled CD4 + CD25 − T cells in a 1:1 ratio for 72 h. ** P < .01, *** P < .001 vs the PBS control group; ## P < .01, ### P < .001 vs the LPS group. (C, F) Proliferative activity of CD4 + CD25 − T cells examined by CFSE staining in mice that received an intra‐abdominal injection of IL‐38 (1 μg per mouse) or PBS (the control group) and that underwent CLP 2 h later. CD4 + CD25 + Tregs were isolated from the spleens of mice that underwent different treatments at 48 h and co‐cultured with CFSE‐labelled CD4 + CD25 − T cells at a 1:1 ratio for 72 h. * P < .05, *** P < .001 vs. the PBS control group; ** P < .01 vs the NC group; ### P < .001 vs the CLP group (n = 6 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Activity Assay, Isolation, Staining, Incubation, Injection, Cell Culture

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Impact of IL‐38 on IL‐2, IL‐4 and IFN‐γ levels in CD4 + CD25 − T cells in co‐culture experiments. CD4 + CD25 − T cells and CD4 + CD25 + Tregs were isolated from murine splenocytes. CD4 + CD25 + Tregs were treated with IL‐38 (50, 100 or 200 ng/mL) and then co‐cultured with CD4 + CD25 − T cells. (A‐F) IFN‐γ, IL‐2 and IL‐4 levels in the supernatants of CD4 + CD25 + Tregs co‐cultured with CD4 + CD25 − T cells after 72 h, based on ELISA. * P < .05, ** P < .01, *** P < .0001 vs the PBS control group; ## P < .01, ### P < .001 vs the LPS group (n = 6 per group). (G‐I) IL‐2, IL‐4 and IFN‐γ levels in the supernatants of CD4 + CD25 + Tregs co‐cultured with CD4 + CD25 − T cells after 72 h. * P < .05, *** P < .01 vs the NC group; # P < .05, ### P < .001 vs the CLP group (n = 6 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Co-Culture Assay, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Blockade of IL‐38 down‐regulated the capacity of IL‐38 to activate CD4 + CD25 + Tregs. CD4 + CD25 − T cells and CD4 + CD25 + Tregs were isolated from murine splenocytes. After pre‐stimulation with IL‐38 at 100 ng/mL, CD4 + CD25 + Tregs were treated for 48 h with anti–IL‐38antibody (500 ng/mL) together with LPS (5 μg/mL). In other experiments, mice were intraperitoneally treated IL‐38 (1 μg per animal) then subjected to CLP 2 h later and finally given anti–IL‐38 antibody intraperitoneally (50 μg per mouse) immediately after CLP, followed 24 h later by a booster dose of 50 μg. (A, B, O, P) Expression of Foxp3 and CD152 at 48 hours performed with flow cytometry. * P < .05, ** P < .01 vs the LPS + IL‐38 group or the CLP + IL‐38 group (n = 6 per group). (C‐F) Levels of IL‐10 and TGF‐β1 in the supernatants based on ELISA. ** P < .01 vs the LPS + IL‐38 group or the CLP + IL‐38 group (n = 6 per group). (G‐L) IFN‐γ, IL‐2 and IL‐4 levels in the supernatants based on ELISA. *** P < .001 vs the LPS + IL‐38 group or the CLP + IL‐38 group (n = 6 per group). (M, N, Q, R) Proliferation of CD4 + CD25 − T cells according to CFSE staining after 48‐h co‐culture with CD4 + CD25 + Tregs. ** P < .01 vs. the LPS + IL‐38 group or the CLP + IL‐38 group (n = 6 per group)

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Isolation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Co-Culture Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Effects of IL‐38 on the 7‐day survival rate of septic mice (A) Mice were treated with IL‐38 at 0.25, 0.5 or 1 μg per mouse and challenged with severe CLP 2 h later. In the control group, an equal volume of PBS was injected intraperitoneally. The results of each treatment group were compared with the results of the CLP + PBS group based on the log‐rank test (n = 25 per group). ** P < .01, *** P < .001 vs the CLP + PBS group. B, Mice received an intra‐abdominal injection of IL‐38 (1 μg per animal) at 2 h before or after severe CLP. * P < .05, *** P < .001 vs the CLP + PBS group (n = 25 per group). C, Mice underwent non‐severe CLP and immediately injected intraperitoneally with anti–IL‐38 antibody (50 μg per mouse, followed by a booster dose of 50 μg 24 h later). Control animals received rat IgG1. *** P < .001 vs. the CLP + IgG1group

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: Interleukin‐38 protects against sepsis by augmenting immunosuppressive activity of CD4 + CD25 + regulatory T cells

doi: 10.1111/jcmm.14902

Figure Lengend Snippet: Impact of CD4 + CD25 + Treg depletion on IL‐38–mediated protection against sepsis CD4 + CD25 + Tregs was isolated from murine splenocytes. (A, B) Flow cytometry plots showed CD4 + CD25 + Tregs in the spleen at 48 h after treatment with anti‐CD25 antibody (PC61) (n = 6 per group). *** P < .001 vs mice treated with IgG1 as a control. C, Survival rates of mice depleted of CD4 + CD25 + Tregs by PC61 and treated with IL‐38 after severe CLP. Groups were compared by the log‐rank test (n = 25 per group). *** P < .001 vs mice treated with IgG1 as a control

Article Snippet: Recombinant murine IL‐38 and anti–IL‐38 antibody were obtained from R&D Systems, Minneapolis, MN.

Techniques: Isolation, Flow Cytometry

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Expression of interleukin (IL)-38 is upregulated in osteoarthritis (OA). a) Representative images of cartilage damage in OA mice assessed by safranin O-fast green staining (n = 10; 200×); b) Osteoarthritis Research Society International (OARSI) scores of cartilage damage in OA mice, *p < 0.05 versus sham-operated mice (n = 10). c) IL-38 level in synovial fluid from OA mice detected by enzyme-linked immunosorbent assay (ELISA). *p < 0.05 versus sham-operated mice at different timepoints (n = 5). d) IL-38 level in the supernatant of chondrocytes after IL-1β treatment detected by ELISA. *p < 0.05 versus control chondrocytes. e) Messenger RNA (mRNA) and protein expressions of IL-38 in cartilage tissues from OA mice (n = 10). *p < 0.05 versus sham-operated mice (n = 10). f) and g) mRNA (f) and protein (g) expression of IL-38 in chondrocytes after IL-1β treatment measured by quantitative reverse transcription polymerase chain reaction and western blot analysis. h) The expression of IL-38 in chondrocytes determined via immunofluorescence (400×). *p < 0.05 versus control chondrocytes. The measurement data were expressed as mean (standard deviation). Comparison between two groups was conducted by independent-samples t -test. The cell experiment was repeated three times independently. Ctr, control.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Staining, Enzyme-linked Immunosorbent Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Western Blot, Immunofluorescence, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Exogenous overexpression of interleukin (IL)-38 attenuates inflammatory response of osteoarthritis (OA) mice. a) Messenger RNA (mRNA) expression of IL-38 in knee joint cartilage tissues from OA mice and sham-operated mice in response to lentivirus vector (LV)-IL-38 or LV-negative control (NC), as determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. b) IL-38 protein level in the knee joint cartilage tissues (left) and synovial fluid (right) from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC as measured using western blot analysis and enzyme-linked immunosorbent assay (ELISA). *p < 0.05 versus sham-operated mice injected with LV-NC; #p < 0.05 versus OA mice injected with LV-NC. c) Levels of inflammation-related factors (IL-6, IL-8, IL-17, IL-22, tumour necrosis factor (TNF)-α, interferon (IFN)-γ, and cartilage oligomeric matrix protein (COMP)) in synovial fluid from OA mice and sham-operated mice in response to LV-IL-38 or LV-NC, tested using ELISA. d) The Osteoarthritis Research Society International (OARSI) score of cartilage damage in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC identified by safranin O-fast green staining. e) Chondrocyte apoptosis in OA mice and sham-operated mice treated with LV-IL-38 or LV-NC detected using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. In panels c) to f), *p < 0.05 versus OA mice with injection of LV-NC. The measurement data were expressed as mean (standard deviation). The cell experiment was repeated three times independently. Comparison between two groups was conducted by independent-samples t -test; n = 10.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Over Expression, Expressing, Plasmid Preparation, Negative Control, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Injection, Western Blot, Enzyme-linked Immunosorbent Assay, Staining, TUNEL Assay, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Long noncoding RNA (lncRNA) H19 increases interleukin (IL)-38 expression through transcription factor TP53. a) Prediction of potential transcription factors regulating IL-38 by PROMO and CIS-BP databases. b) Messenger RNA (mRNA) expression of ETS1, TP53, GATA2, GATA1, ELK1, MAZ, and ETS2 in osteoarthritis (OA) mice and chondrocytes. *p < 0.05 versus sham-operated mice or control chondrocytes. c) TP53 enrichment of lncRNAs GAS5, MEG3, TUG1, MALAT1, and H19 determined by RNA binding protein immunoprecipitation (RIP) assay. Results are normalized to immunoglobulin G (IgG). *p < 0.05 versus anti-IgG. d) mRNA expression of lncRNA H19 in OA mice (n = 10) and chondrocytes. *p < 0.05 versus sham-operated mice or control chondrocytes. e) Relative luciferase activity of IL-38 promoter in OA chondrocytes after various treatments. *p < 0.05 versus OA chondrocytes treated with oe-negative control (NC), #p < 0.05 versus OA chondrocytes treated with sh-NC. f) Relative enrichment of lncRNA H19 by TP53 determined by RIP assay. *p < 0.05 versus anti-IgG. g) Relative enrichment of IL-38 by TP53 in OA chondrocytes determined by chromatin immunoprecipitation (ChIP) assay. *p < 0.05 versus anti-IgG. h) Transfection efficiency in OA chondrocytes detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). *p < 0.05 versus OA chondrocytes treated with both oe-NC and sh-NC. i) IL-38 level in OA chondrocytes after various treatments. *p < 0.05 versus OA chondrocytes co-treated with oe-NC and sh-NC. j) Transfection efficiency in OA mice detected by RT-qPCR. *p < 0.05 versus OA mice treated with lentivirus vector (LV)-NC. k) IL-38 levels in OA mice after various treatments. *p < 0.05 versus OA mice treated with LV-NC. l) Pearson analysis for the expression of lncRNA H19 and IL-38 in knee joint cartilage tissues from OA mice. The measurement data were expressed as mean (standard deviation). Comparison between two groups was conducted by independent-samples t -test. Comparison among multiple groups was conducted by one-way analysis of variance, followed by Tukey’s post hoc test; n = 10. The cell experiment was repeated three times independently. Ctr, control.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Control, RNA Binding Assay, Immunoprecipitation, Luciferase, Activity Assay, Negative Control, Chromatin Immunoprecipitation, Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Elevated long noncoding RNA (lncRNA) H19 exerts anti-inflammation effects on osteoarthritis (OA) by promoting interleukin (IL)-38, tumour protein p53 (TP53), and IL-36 receptor (IL-36R). a) Transfection efficiency in knee joint cartilage tissues from OA mice detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). b) Levels of inflammation-related factors (IL-6, IL-8, IL-17, IL-22, TNF-α, interferon (IFN)-γ, and cartilage oligomeric matrix protein (COMP)) in synovial fluid from OA mice with various treatments. c) The Osteoarthritis Research Society International (OARSI) score of cartilage damage in OA mice with various treatments identified by safranin O-fast green staining. d) Chondrocyte apoptosis after various treatments determined by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay. *p < 0.05 versus OA mice treated with lentivirus vector (LV)-negative control (NC); #p < 0.05 versus OA mice treated with LV-oe-H19. The cell experiment was repeated three times independently. The measurement data were expressed as mean (standard deviation). One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s post hoc test; n = 10.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Transfection, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Staining, TUNEL Assay, Plasmid Preparation, Negative Control, Standard Deviation, Comparison

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: Long noncoding RNA (lncRNA) H19/interleukin (IL)-38 axis involving IL-36 receptor (IL-36R) harbours the anti-inflammatory property in osteoarthritis (OA) in vitro. a) Binding between mouse IL-38 and IL-36R in peripheral blood mononuclear cells (PBMCs) analyzed by receptor-binding assay. b) Binding between IL-38 and IL-36R in PBMCs detected by co-immunoprecipitation assay. c) Transfection efficiency detected by quantitative reverse transcription polymerase chain reaction. d) Levels of pro-inflammatory factors in PBMCs after various treatments. In panels c) and d), *p < 0.05 versus co-treatment of oe-negative control (NC) and sh-NC; #p < 0.05 versus co-treatment of oe-H19 and sh-NC. The measurement data were expressed as mean (standard deviation). One-way analysis of variance was used for data comparison among multiple groups, followed by Tukey’s post hoc test. Comparison of IL-38 expression among multiple groups was analyzed by two-way analysis of variance, followed by Bonferroni correction. The cell experiment was repeated three times independently. GFP, green fluorescence protein; IFN, interferon; OD, optical density.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: In Vitro, Binding Assay, Reporter Assay, Co-Immunoprecipitation Assay, Transfection, Reverse Transcription, Polymerase Chain Reaction, Negative Control, Standard Deviation, Comparison, Expressing, Fluorescence

Journal: Bone & Joint Research

Article Title: Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor

doi: 10.1302/2046-3758.118.BJR-2021-0188.R1

Figure Lengend Snippet: The regulatory mechanism of the long noncoding RNA (lncRNA) H19/tumour protein p53 (TP53)/interleukin (IL)-38 axis involved in osteoarthritis (OA). lncRNA H19 was upregulated in OA. H19 upregulated the expression of IL-38 by recruiting TP53 to the IL-38 promoter region, which promoted binding of IL-38 with IL-36 receptor and inhibited the inflammatory response in OA. IL-36R, interleukin-36 receptor; mRNA, messenger RNA.

Article Snippet: Further, recombinant mouse IL-38 (R&D Systems) was diluted with PBS containing 0.5% BSA and 0.05% Tween-20, and then incubated in a coated/blocked ELISA plate at 4°C overnight.

Techniques: Expressing, Binding Assay