Journal: Arthritis and rheumatism

Article Title: T lymphocytes and muscle condition act like seeds and soil in a murine polymyositis model.

doi: 10.1002/art.34629

Figure Lengend Snippet: Figure 2. Selective transfer of myositis to CFA/Toll-like receptor (TLR) ligand–treated legs. A, Lymph node (LN) cells from mice with C protein–induced myositis (CIM) were stimulated with C protein fragment–pulsed mature bone marrow–derived dendritic cells (BMDCs) or untreated mature BMDCs (no antigen) for 3 days. Their proliferation was determined by 3H-thymidine incorporation (top). Interferon- (IFN) in the culture supernatants was quantified by enzyme-linked immunosorbent assay (bottom). Values are the mean SD of 3 independent experiments. P 0.01. B, LN cells from mice with CIM stimulated with interleukin-2 (IL-2) and C protein fragment–pulsed mature BMDCs were transferred to 5 naive mice with their right hind leg footpads treated with CFA and their left hind leg footpads treated with IFA. Six naive mice were treated with CFA on their hind leg footpads without adoptive transfer. Myositis of the bilateral femoral muscles was histologically assessed 14 days after the transfer. Horizontal bars indicate the mean. P 0.05. C, Upon adoptive transfer of LN cells from mice with CIM stimulated with IL-2 and C protein fragment–pulsed mature BMDCs, the legs of the recipient mice were treated with the TLR ligands poly(I-C), LPS, and CpG-containing oligonucleotide (CpG ODN; CpG DNA). CFA and IFA were included as positive and negative controls, respectively. The incidence of myositis resulting from the transfer is shown. Each group included 5 mice. See Figure 1 for other definitions.

Article Snippet: The culture supernatants were examined for the concentration of IFN with an enzymelinked immunosorbent assay (ELISA) kit (Mouse IFN-gamma DuoSet; R&D Systems).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Muscles

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: CTRP3 inhibits the differentiation of Th17 cells in an autocrine manner. (A) The relative expression of C1qtnf3 mRNA in different Th subsets was determined by real-time PCR, and the expression levels are shown relative to that of Th0 cells. Th0, Th1, Th2 and Th17 cells were prepared as described in the Materials and Methods section. (B) WT or C1qtnf3 –/– (KO) naïve CD4 + T cells were cultured under Th17-polarizing conditions for 4 days (n = 4 each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The numbers in each panel indicates percentage of IL-17 + CD4 + T cells in total CD4 + T cells (2 left panels). The content of IL-17 + CD4 + T cells in total CD4 + T cells (%) (center), and IL-17 concentrations (ng/ml) in culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, *** p < 0.001. Student’s t -test. (C) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of recombinant CTRP3 (50, 100 and 200 ng/ml) for 4 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IL-17 + CD4 + T cells (left), and the proportion of IL-17 + CD4 + T cells is shown in the center panel. IL-17 concentration in the culture supernatant was determined by ELISA (right). Average and SEM are shown. ** p < 0.01, *** p < 0.001. Student’s t -test. (D) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th1-polarizing conditions for 3 days (n = 4 wells each). Intracellular IFN-γ expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates the percentage of IFN-γ + CD4 + T cells in total CD4 T cells (left). The population of IFN-γ + CD4 + T cells (center). IFN-γ concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. Student’s t -test. (E) The expression of Adipor1 and Adipor2 mRNA in different Th subsets was determined by real-time PCR and relative expression levels to that in Th0 cells are shown. (F) C1qtnf3 –/– naïve CD4 + T cells were cultured with recombinant CTRP3 (200 ng/ml) under Th17-polarizing conditions in the absence (-) or presence of AdipoR1 blocker (R1 block, 10 μg/ml) or AdipoR2 blocker (R2 block, 10 μg/ml) for 4 days (n = 4 each). Intracellular IL-17 expression was evaluated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17 + CD4 + T cells (left). The IL-17 + CD4 + T cell content (center). IL-17 concentration in the culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, ** p < 0.01. Student’s t -test. (G) The effect of AdipoR1 blocker and AdipoR2 blocker on the regulatory effects of CTRP3 on Th17 cell gene expression was examined. In vitro differentiated C1qtnf3 –/– Th17 cells (1 x 10 5 cells in 200 μl/96 well) were treated with CTRP3 (200 ng/ml) in the presence or absence of AdipoR1 blocker (10 μg/ml, R1 block) or AdipoR2 blocker (10 μg/ml, R2 block), and the expression of Ampka , Ppara , Rorc , Stat3 and Mtor mRNA was determined by real-time PCR. The relative expression levels to that of Gapdh are shown. These data are the average from three independent experiments. Average and SEM are shown. * p < 0.05, ** p < 0.01. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Blocking Assay, Gene Expression, In Vitro

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: AdipoRon suppresses Th17 cell differentiation via AdipoR2. (A) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (0, 1, 2 and 3 μg/ml) for 5 days (n = 4 wells each). Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The number in each panel indicates percentage of IL-17 + CD4 + T cells (2 left panels). The population of IL-17 + CD4 + T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. ** p < 0.01 and *** p < 0.001. Student’s t -test. (B) C1qtnf3 –/– naïve CD4 + T cells were cultured under Th17-polarizing conditions in the absence or presence of AdipoRon (1 μg/ml, Ron) for 4 days (n = 3 wells each), and the effect of AdipoR1 (10 μg/ml, R1) or AdipoR2 blocker (10 μg/ml, R2) on the Th17 cell differentiation was examined. Intracellular IL-17 expression was estimated by flow cytometry after PMA/ionomycin stimulation. The Number in each panel indicates percentage of IL-17 + CD4 + T cells (4 left panels). The population of IL-17 + CD4 + T cells (center). IL-17 concentrations in culture supernatant determined by ELISA (right). Average and SEM are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Cell Differentiation, Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: Development of EAE is exacerbated in C1qtnf3 –/– mice associated with an augmented Th17 cell differentiation. (A–D) Incidence of affected mice (A) , onset time (B) , disease score time course (C) and mean maximum disease score (D) are shown after induction of EAE (11 mice for each group). Average and SEM are shown. * p < 0.05 and *** p < 0.001. χ 2 -test. Mann-Whitney U -test. Student’s t -test. (E, F) Tissue sections of the spinal cord were stained with H&E (E) and LFB (F) at day 28 after induction of EAE. (G, H) CD4 + T cells (G) , CD4 + IL-17 + and CD4 + IFN-γ + T cells (H) in the whole spinal cord from WT and C1qtnf3 –/– (KO) mice at day 21 after immunization were determined by flow cytometry after PMA/ionomycin stimulation (WT, KO: n = 5 each). The numbers in each panel indicate the percentage of IL-17 + , IFN-γ + , and IL-17 + IFN-γ + T cells ( H , 2 left panels). The population of IL-17-single positive, IFN-γ-single positive and IL-17-, IFN-γ-double positive T cells (H, 3 right panels). Average and SEM are shown. *** p < 0.001. Student’s t -test. (I, J) T cell recall response against the MOG peptide. Seven days after MOG/CFA-immunization, LN cells were isolated from WT and C1qtnf3 –/– (KO) mice and re-stimulated with MOG 35-55 (0, 25, 50 μg/ml) for 72 h (n = 5 each). Then, the proliferative response was measured by [ 3 H]TdR incorporation (I) . IL-17 ( J , left) and IFN-γ ( J , right) concentrations in the culture supernatant were determined by ELISA. Average and SEM are shown. * p < 0.05. Student’s t -test. (K) C1qtnf3 –/– 2D2 T cells and C1qtnf3 -/- BMDCs were cultured with 5 μg/ml MOG 35-55 in the presence or absence of CTRP3 (0, 1, 10, 100 ng/ml) for 5 days, and Th17 cell content (left), and IL-17 (center) and IFN-γ (right) concentrations in the culture supernatant were determined (n = 4 wells each). Average and SEM are shown. * p < 0.05 and ** p < 0.01. Student’s t -test. (L) WT or C1qtnf3 -/- 2D2 T cells were cultured with WT or C1qtnf3 -/- BMDCs in the presence of 5 μg/ml MOG 35-55 for 5 days (n = 3 each), and IL-17 (left) and IFN-γ (right) concentrations in the culture supernatant were determined. Average and SEM are shown. * p < 0.05, ** p < 0.01 and *** p < 0.001. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Cell Differentiation, MANN-WHITNEY, Staining, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: The CTRP3-AdipoR2 Axis Regulates the Development of Experimental Autoimmune Encephalomyelitis by Suppressing Th17 Cell Differentiation

doi: 10.3389/fimmu.2021.607346

Figure Lengend Snippet: DC differentiation is normal in C1qtnf3 -/- mice. (A) C1qtnf3 mRNA in immune cells was determined by real-time PCR, and the relative expression levels to that in B cells are shown in left panel. C1qtnf3 mRNA expression in DCs after stimulation with zymosan or LPS was determined by real-time PCR, and the relative expression levels to that in cells without stimulation (-) are shown in right panel. (B, C) Effect of CTRP3 on the differentiation and proliferation of BMDCs. BMDCs were differentiated from WT or C1qtnf3 –/– (KO) BMCs (B) , or from C1qtnf3 –/– BMCs in the presence of indicated concentrations of CTRP3 (C) . Then, viable cell number was counted at day 8 by using Cell Counting Kit-8. 4 wells for each. Average and SEM are shown. Student’s t -test. (D) WT or C1qtnf3 -/- (KO) BMCs were cultured under BMDC-differentiation conditions, and the expression of CD11c, CD40, CD80 and CD88 was determined by flow cytometry at day 8. (E) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 –/– (KO) BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml). Four wells for each. Average and SEM are shown. Student’s t -test. (F) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 –/– BMDCs were measured by ELISA after stimulated with zymosan (0, 40 and 80 μg/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. Student’s t -test. (G) IL-6 and TNF-α concentration in the culture supernatant of WT or C1qtnf3 –/– (KO) BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml). Four wells for each. Average and SEM are shown. * p < 0.05, *** p < 0.001. Student’s t -test. (H) IL-6 and TNF-α concentration in the culture supernatant of C1qtnf3 –/– BMDCs were measured by ELISA after stimulated with LPS (0, 10 and 20 ng/ml) with/without CTRP3. Four wells for each. Average and SEM are shown. * p < 0.05. Student’s t -test. All data were reproduced in another independent experiment with similar results.

Article Snippet: IL-17A, IFN-γ, IL-6 and TNF-α in culture supernatants were measured using mouse IL-17 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IFN-γ ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), mouse IL-6 ELISA MAX standard (R&D Systems, Minneapolis, MN, USA) and mouse TNF-α ELISA MAX standard (R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Cell Counting, Cell Culture, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

doi: 10.1074/jbc.m113.534396

Figure Lengend Snippet: FIGURE 3. Changes in IL-33 (A, B, C, and E), IFN- (D), and IL-4 (F) protein levels with mean S.D. as measured by ELISA in triplicate cultures. Each experiment was repeated on 2–5 separate occasions with similar results. A and B, NIH3T3 fibroblasts were infected with AdV-RSV-IL33 (A), AdV-CMV-IL33 (B), or AdV-NULL control, as indicated. After 48 h, cells were treated with the indicated concentrations of rIFN- for an additional 72 h before the assays. Data for cell lysates are shown; IL-33 was undetectable in cell culture supernates. C–F, primary NMLFs were infected with AdV encoding the indicated proteins, and cell lysates (black bars) and supernates (gray bars) were collected and analyzed 48 h post-infection. Asterisks mark significant (p 0.05) differences in the levels of indicated cytokines between dual and single gene delivery.

Article Snippet: The solid tissue was separated by centrifugation, and the supernatant was used for ELISA of mouse IL-33 and mouse IFN- (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Control, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

doi: 10.1074/jbc.m113.534396

Figure Lengend Snippet: FIGURE 5. ELISA of whole lung homogenates for the indicated cytokines (mean S.D.) on day 7 (gray bars) and day 14 (black bars) after intratracheal instillation of the corresponding AdVs. The levels of statistical significance are indicated only for selected pairwise comparisons to preserve clarity. In A and C, the indicated differences refer to both days 7 and 14. The differences between AdV-IFN and AdV-IL33&IFN (B) or AdV-IL4 and AdV-IL33&IL4 (D) were significant on day 14 but not day 7. This experiment was repeated on three different occasions using 3–5 animals/group/time point, with similar results.

Article Snippet: The solid tissue was separated by centrifugation, and the supernatant was used for ELISA of mouse IL-33 and mouse IFN- (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

doi: 10.1074/jbc.m113.534396

Figure Lengend Snippet: FIGURE 7. Changes in IL-33 protein level (mean S.D.) in cell lysates measured by ELISA in triplicate cultures. Each experiment was repeated on at least six independent occasions, with similar results. A–C, NIH3T3 fibroblasts were treated with specific inhibitors of caspase-1 (Z-WEHD-FMK), -3 (Z-DEVD-FMK), or -8 (Z-IETD-FMK) for 4 h and then infected with AdV constructs encoding IL33, IFN, or IL33&IFN and incubated for an additional 72 h (black bars). D, NIH3T3 fibroblast cultures, 48 h after infection with AdV-IL33, were treated with caspase-1 (Z-WEHD-FMK), -3 (Z-DEVD-FMK), or -8 (Z-IETD-FMK) inhibitors for 4 h and then activated with rIFN- (10 ng/ml) for an additional 72 h (black bars). Similarly processed cultures without rIFN- activation were used as controls (gray bars).

Article Snippet: The solid tissue was separated by centrifugation, and the supernatant was used for ELISA of mouse IL-33 and mouse IFN- (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Construct, Incubation, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

doi: 10.1074/jbc.m113.534396

Figure Lengend Snippet: FIGURE 8. A, changes in IL-33 protein levels measured by ELISA in cell lysates in triplicate cultures (mean S.D.). Each experiment was repeated on three separate occasions with similar results. NIH3T3 fibroblasts were treated with nifuroxazide (300 M) (black bars) for 1 h, infected with AdV constructs encoding IL-33, IFN-, IL33&IFN, or NULL, and incubated for an additional 72 h. Cells without nifuroxazide treatment but with delivery of the same gene were used as a control (gray bars). B, luciferase assay of NIH3T3 cells transfected with the STAT1-responsive promoter-luciferase reporter construct (black bars), positive control (gray bars) (a constitutive luciferase-expressing construct), and negative control (white bars) (a non-expressing construct) and treated with 100 ng/ml rIFN-, 300 M nifuroxazide (NF), or their combination, as indicated, for 24 h. C, Western blot analysis of STAT1 (upper gel) and -actin (lower gel) in cell culture lysates of NIH3T3 fibroblasts transfected with control scrambled (lane 1) or STAT1 siRNA (lane 2). D, NIH3T3 cells were transfected with 250 M siRNA against STAT1 (black bars) or non-targeting scrambled siRNA (white bars). At 48 h post-siRNA transfection, cells were infected with AdV constructs encoding IL33, IFN-, IL33&IFN, or NULL and incubated for an additional 72 h. Cells without previous siRNA transfection but with delivery of the same gene were used as a control (gray bars). E, STAT1/ primary mouse lung fibroblasts were infected with IL-33 alone, IL33&IFN, or IL-33 with subsequent treatment with rIFN- (10 ng/ml). NMLFs were used as a control. The difference in IL-33 expression levels between cells from STAT1/ and WT mice were significant (p 0.05) in all groups, whereas the difference between STAT1/ cells in these groups were not significant (p 0.05).

Article Snippet: The solid tissue was separated by centrifugation, and the supernatant was used for ELISA of mouse IL-33 and mouse IFN- (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Construct, Incubation, Control, Luciferase, Transfection, Positive Control, Expressing, Negative Control, Western Blot, Cell Culture

Journal: Journal of Biological Chemistry

Article Title: IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism

doi: 10.1074/jbc.m113.534396

Figure Lengend Snippet: FIGURE 9. A, Western blot analysis of LMP2 (upper gel) and -actin (lower gel) in cell culture lysates of NIH3T3 fibroblasts infected with AdV-NULL (lane 1), AdV-IFN (lane 2), AdV-IL33 (lane 3), or AdV-IL33&IFN (lane 4). B, Western blot analysis of LMP2 (upper gel) and -actin (lower gel) of whole lung homogenates after intratracheal instillation of AdV-NULL (lane 1), AdV-IFN (lane 2), AdV-IL33 (lane 3), AdV-IL33&IFN (lane 4). C, Western blot analysis of LMP2 (upper gel) and -actin (lower gel) in cell culture lysates of NIH 3T3 fibroblasts transfected with control scrambled (lane 1) or LMP2 siRNA (lane 2) and treated with 100 ng/ml of rIFN-. D, changes in IL-33 protein level S.D. measured in cell culture lysates by ELISA in triplicate cultures; each experiment was repeated twice with similar results. NIH3T3 fibroblasts were transfected with 500 M siRNA against LMP2 or non-targeting scrambled siRNA. At 48 h post-transfection cells were infected with AdV constructs encoding IL-33, IFN-, or IL-33 and IFN- genes and incubated for an additional 72 h. Cells without previous transfection with siRNAs but with delivery of corresponding genes were used as a control.

Article Snippet: The solid tissue was separated by centrifugation, and the supernatant was used for ELISA of mouse IL-33 and mouse IFN- (R&D Systems).

Techniques: Western Blot, Cell Culture, Infection, Transfection, Control, Enzyme-linked Immunosorbent Assay, Construct, Incubation