Journal: Scientific Reports

Article Title: The protective effect of the anti-Toll-like receptor 9 antibody against acute cytokine storm caused by immunostimulatory DNA

doi: 10.1038/srep44042

Figure Lengend Snippet: ( a , c , e ) RAW264.7 cells ( a ), BM-Macs( c ) and BM-cDCs ( e ) were treated with medium, lipid A (100 ng/ml), CpGB (500 nM), IFN-β (1000 U/ml) or TNF-α (25 ng/ml). Twenty-four hours later, cells were subjected to cell surface (upper panel) or membrane-permeabilized staining (lower panel). Grey histograms show staining with isotype control. Black and red open histograms show staining with NaR9 before and after treatment. ( b , d , f ) RAW264.7 cells ( b ), BM-Macs ( d ) and BM-cDCs ( f ) were pre-treated with medium, IFN-β (1000 U/ml) or TNF-α (25 ng/ml). Twenty-four hours later, the cells were stimulated with the indicated concentration of CpGB or CpGA. Twenty-four hours after stimulation with CpGB or CpGA, culture supernatants were collected, and the concentration of RANTES ( b ) or IL-6 ( d , f ) was determined by ELISA. The results are represented by the mean value and the s.d. from triplicate wells. These experiments were repeated at least twice, and representative data are shown.

Article Snippet: ELISA Sets (eBiosciences), and IFN-α was measured using a VeriKine ELISA Kit (PBL Assay Science, Piscataway, NJ, USA).

Techniques: Membrane, Staining, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: The protective effect of the anti-Toll-like receptor 9 antibody against acute cytokine storm caused by immunostimulatory DNA

doi: 10.1038/srep44042

Figure Lengend Snippet: ( a–c ) BM-Macs ( a ), BM-cDCs ( b ) and BM-pDCs ( c ) were stimulated with CpGB or CpGA. As a control, cells were stimulated by loxoribine or poly U with DOTAP. Four hours before stimulation, the cells were treated with NaR9 or isotype-matched control mAb (IgG2a) at the indicated concentrations. Supernatant was collected after 24 h of stimulation, and cytokine production was evaluated by ELISA. ( d , e ) BM-Macs ( d ) and BM-cDCs ( e ) were pre-treated with medium or IFN-β (1000 U/ml) for 24 h. The cells were treated with NaR9 or isotype-matched control mAb (IgG2a) at the indicated concentrations for 4 h and stimulated by 50 nM of CpGB. Twenty-four hours after stimulation with CpGB, culture supernatants were collected, and the concentration of IL-6 was determined by ELISA. The results are represented by the mean value and the s.d. from triplicated wells. These experiments were repeated at least 3 times ( a – c ) or twice ( d , e ), and representative data are shown.

Article Snippet: ELISA Sets (eBiosciences), and IFN-α was measured using a VeriKine ELISA Kit (PBL Assay Science, Piscataway, NJ, USA).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Scientific Reports

Article Title: The protective effect of the anti-Toll-like receptor 9 antibody against acute cytokine storm caused by immunostimulatory DNA

doi: 10.1038/srep44042

Figure Lengend Snippet: ( a ) C57BL/6 mice were intraperitoneally administered with anti-TLR9 or isotype-matched IgG2a mAb (25 mg/kg) 15 h before injection of CpGB (500 nmol/kg) and D-(+)-galactosamine (1 g/kg). The percentage of mice that survived the treatments is shown. Each group has 15 mice. ( b ) Blood was collected from mice at indicated time points after injection of CpGB and D-(+)-galactosamine. Serum TNF-α and IL-12p40 levels were measured by ELISA. Bars in graphs indicate the mean values. Each group has 6 mice. Statistical analysis was performed using a Student’s t-test: * p < 0.05.

Article Snippet: ELISA Sets (eBiosciences), and IFN-α was measured using a VeriKine ELISA Kit (PBL Assay Science, Piscataway, NJ, USA).

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Chinese Medicine

Article Title: Microbiota-driven tryptophan metabolism and AhR triggered intestinal stem cell differentiation: mechanisms of huangqin decoction in ulcerative colitis repair

doi: 10.1186/s13020-025-01302-y

Figure Lengend Snippet: Blockade of AhR signalling compromises the ameliorative effects of HQD in colitis mice. A Body weight changes. B The DAI score. C The colon length of mice. D The intestinal permeability. E H&E staining. F AhR and CYP1A1 protein expression level. G-I AhR, CYP1A1, and IL-22 mRNA expression. J The content of IL-22 was evaluated using ELISA. Data were presented as mean ± SD (n = 3–6). ## P < 0.01, ### P < 0.001, #### P < 0.0001, vs. Normal group. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs. DSS group. ▲ P < 0.05, ▲▲▲ P < 0.001, ▲▲▲▲ P < 0.0001, vs. HQD group

Article Snippet: The enzyme-linked immunosorbent assay (ELISA) kit of IL-22 (E-EL-M2446) was purchased from Elabscience (Wuhan, China).

Techniques: Permeability, Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: IL-22BP protein and mRNA levels in monocyte-derived DCs. (A) (Upper left) Diagram showing exons with the primer-binding positions for IL22RA2 variant discrimination strategy. (Lower left) Expression of IL22RA2v1, v2 , and v3 in moDC after 6 days of culture in differentiation medium (DM) ± AM580, a retinoic acid receptor agonist, by RT-PCR. IL22RA2v2 expression vector was used as positive control for IL22RA2v2 expression. (Upper right) Expression of IL22RA2 variants in moDC following 6 days of culture in DM ± AM580 by RT-qPCR using the isoform-specific primers indicated in the left diagram (mean ± SEM; n = 3) relative to the housekeeping gene GAPDH . (B) Effect of different maturation stimuli (CpG and LPS + IFN-γ or Poly I:C) on IL22RA2, IL12B , and IL6 expression in moDC after 6 days of culture in DM by RT-qPCR using Taqman probes relative to the housekeeping gene ACTB . Extent of maturation is represented as percentage of CD83 + fluorescence by flow cytometry. Data showing representative experiment of 3 performed. (C) IL-22BP secretion by moDC cultured in DM detected by ELISA corresponds to IL22RA2 mRNA expression levels analyzed by RT-qPCR using Taqman probes and both increase over the cultivation period (mean ± SEM, n = 2). (D) Cell lysates (CL) of moDCs and conditioned media (CM) were harvested after 6 days in DM ± AM580 and immunoblotted for detection of IL-22BP (IL-22BP antibody 1) using tubulin as a loading control, IL22RA2 mRNA expression relative to the mean of the housekeeping genes GAPDH and ACTB was measured by RT-qPCR using pre-designed SYBR Green primers from the same cells. Representative experiment out of three performed. All primers used are listed in Supplementary Table .

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Derivative Assay, Binding Assay, Variant Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Positive Control, Quantitative RT-PCR, Fluorescence, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, SYBR Green Assay

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments.

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Confocal Microscopy, Marker, Recombinant, Generated, Subcloning

Journal: Frontiers in Immunology

Article Title: Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

doi: 10.3389/fimmu.2018.02934

Figure Lengend Snippet: Silencing of IL22RA2 in immature moDCs reduces GRP78 expression. (A) CD14 + monocytes were stained with DAPI (blue), trans-GOLGI (red) and IL-22BP (green, IL-22BP Ab 4). (B) Overall (best coverage, BC) and isoform-specific IL22RA2 expression levels in classical, intermediate and non-classical monocyte subpopulations (top) from four healthy donors (average ± SEM). Percentages of IL-22BP + monocytes found in each subpopulation by immunofluorescence microscopy (bottom) (mean ± SEM; n = 4; * p < 0.05, Kruskal-Wallis test for comparison of differences in IL22RA2 mRNA levels or IL-22BP + counts in the three subpopulations). (C) IL22RA2 expression levels in classical and intermediate immature moDCs at day 6 of cultivation. moDCs derived from non-classical monocytes showed high levels of cell death and had not experienced any changes in the shape of the cells. High Ct values of HK gene HPRT1 in qPCR compromised correct interpretation of IL22RA2 levels in this subgroup, which was therefore excluded from the graph. (D) Fold change relative to non-targeting siRNA control of IL22RA2 expression in CD14 + monocyte.-derived immature DCs on day 5 of cultivation following IL22RA2 silencing with Viromer Green at two concentrations of siRNA. (E) Silencing as described in (D) was not variant specific and both IL22RA2v1 and v2 were silenced to similar extent. (F) Scheme comparing efficiencies of four distinct silencing strategies on suppression of IL22RA2 mRNA (qPCR) and intracellular IL-22BP (ELISA) in immature CD14 + moDCs using 100 nM IL22RA2 siRNA or non-targeting siRNA in combination with Viromer Green. S, silencing, H, harvest. Bar diagrams represent fold change relative to non-targeting siRNA. (G) Effect of IL22RA2 silencing on GRP78 and ERp44 mRNA levels in CD14 + monocyte-derived immature DCs from healthy donors using silencing strategy B in (F) . CD14 + monocytes were cultivated in DM medium in the absence or presence of AM580 starting on day 0 up to the day of harvest. Average of fold change (±SEM) relative to non-silencing control over 6 (control) and 5 (AM580) independent measurements relative to the housekeeping gene HPRT1. * p < 0.03 by Wilcoxon test.

Article Snippet: For confocal immmuofluorescence microcroscopy, we used the following antibodies: anti-IL22RA2 (R&D; AF1087 and BAF1087, 1:20 each), anti-IL22RA2 (Proteintech; 66190-1-Ig), anti-TGOLN (Atlas; HPA012723-100ul, 1:500), anti-ERp72 (Enzo; ADI-SPS-720-F; 1:500), donkey anti-goat A647 (Jackson ImmunoResearch; 705-605-147; 1:500), Streptavidin DyLight 488 (Tebu-bio; 039S000-41; 1:4000), donkey anti-rabbit A647 (Jackson Immunoresearch; 711-605-152; 1:500), as well as DAPI (Sigma; 10 μg/ml).

Techniques: Expressing, Staining, Immunofluorescence, Microscopy, Derivative Assay, Variant Assay, Enzyme-linked Immunosorbent Assay