recombinant mouse il 23  (Thermo Fisher)


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    Name:
    Mouse IL 23 Recombinant Protein
    Description:
    Mouse IL 23 Recombinant Protein for ELISA Ctrl FN
    Catalog Number:
    14-8231-63
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    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher recombinant mouse il 23
    MaR1 inhibits <t>IL-23</t> injection-induced psoriatic skin inflammation. ( a ) Ear swelling response. Ears swelling was measured from Day 0–16 upon topical application of vehicle or MaR1 (100 ng) 30 min before intradermal injection of 500 ng recombinant mouse IL-23 (n = 3) every other day. The mean ear thickness in both ears was calculated in 3 mice. ( b ) Hematoxylin and eosin-stained sections. Ear skin was collected on day 16. Bar, 200 μm. ( c ) Histological examination of epidermal thickness (n = 3). ( d ) The number of γδTCR mid+ and intracellular IL-17A + cells in the skin (n = 4). Ear skin was collected 24 h after IL-23 injection for two consecutive days and subjected to flow cytometry. ( e ) The number of CD4 + and intracellular IL-17A + cells in the skin (n = 4). Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P
    Mouse IL 23 Recombinant Protein for ELISA Ctrl FN
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    Images

    1) Product Images from "Maresin-1 suppresses imiquimod-induced skin inflammation by regulating IL-23 receptor expression"

    Article Title: Maresin-1 suppresses imiquimod-induced skin inflammation by regulating IL-23 receptor expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23623-9

    MaR1 inhibits IL-23 injection-induced psoriatic skin inflammation. ( a ) Ear swelling response. Ears swelling was measured from Day 0–16 upon topical application of vehicle or MaR1 (100 ng) 30 min before intradermal injection of 500 ng recombinant mouse IL-23 (n = 3) every other day. The mean ear thickness in both ears was calculated in 3 mice. ( b ) Hematoxylin and eosin-stained sections. Ear skin was collected on day 16. Bar, 200 μm. ( c ) Histological examination of epidermal thickness (n = 3). ( d ) The number of γδTCR mid+ and intracellular IL-17A + cells in the skin (n = 4). Ear skin was collected 24 h after IL-23 injection for two consecutive days and subjected to flow cytometry. ( e ) The number of CD4 + and intracellular IL-17A + cells in the skin (n = 4). Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P
    Figure Legend Snippet: MaR1 inhibits IL-23 injection-induced psoriatic skin inflammation. ( a ) Ear swelling response. Ears swelling was measured from Day 0–16 upon topical application of vehicle or MaR1 (100 ng) 30 min before intradermal injection of 500 ng recombinant mouse IL-23 (n = 3) every other day. The mean ear thickness in both ears was calculated in 3 mice. ( b ) Hematoxylin and eosin-stained sections. Ear skin was collected on day 16. Bar, 200 μm. ( c ) Histological examination of epidermal thickness (n = 3). ( d ) The number of γδTCR mid+ and intracellular IL-17A + cells in the skin (n = 4). Ear skin was collected 24 h after IL-23 injection for two consecutive days and subjected to flow cytometry. ( e ) The number of CD4 + and intracellular IL-17A + cells in the skin (n = 4). Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P

    Techniques Used: Injection, Recombinant, Mouse Assay, Staining, Flow Cytometry, Cytometry

    MaR1 treatment downregulates and internalizes IL-23 receptors in an in vitro skin assay. The frequencies of γδTCR mid+ and intracellular IL-17A + cells in IL-23 stimulated skin cell suspensions (n = 4). Skin cell suspensions were cultured with recombinant IL-23 with vehicle or MaR1 (10 nM) stimulation for 24 h, and the number of γδTCR mid+ and intracellular IL-17A + cells were analyzed by flow cytometry. ( b ) The number of CD4 + and intracellular IL-17A + cells in in IL-23 stimulated skin cell suspensions (n = 4). ( c , d ) The mean fluorescence intensity (MFI) of IL-23R expression on γδTCR mid+ ( c ) and CD4 + cells ( d ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 24 h and IL-23R expression was measured by flow cytometry. ( e , f ) The MFI of RORγt expression on γδTCR mid+ ( e ) and CD4 + cells ( f ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 3 h and intracellular RORγt expression was measured by flow cytometry. ( g , h ) The MFI of IL-23R expression on γδTCR mid+ ( g ) and CD4 cells ( h ) with internalization inhibitors. Skin suspension cells were cultured with vehicle or MaR1 (10 nM) with nocodazole (10 μM), Pit stop2 (20 μM), nystatin (50 μg/mL), and chlorprozine (25 μg/mL) for 1 h and IL-23R expression was measured by flow cytometry. Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P
    Figure Legend Snippet: MaR1 treatment downregulates and internalizes IL-23 receptors in an in vitro skin assay. The frequencies of γδTCR mid+ and intracellular IL-17A + cells in IL-23 stimulated skin cell suspensions (n = 4). Skin cell suspensions were cultured with recombinant IL-23 with vehicle or MaR1 (10 nM) stimulation for 24 h, and the number of γδTCR mid+ and intracellular IL-17A + cells were analyzed by flow cytometry. ( b ) The number of CD4 + and intracellular IL-17A + cells in in IL-23 stimulated skin cell suspensions (n = 4). ( c , d ) The mean fluorescence intensity (MFI) of IL-23R expression on γδTCR mid+ ( c ) and CD4 + cells ( d ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 24 h and IL-23R expression was measured by flow cytometry. ( e , f ) The MFI of RORγt expression on γδTCR mid+ ( e ) and CD4 + cells ( f ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 3 h and intracellular RORγt expression was measured by flow cytometry. ( g , h ) The MFI of IL-23R expression on γδTCR mid+ ( g ) and CD4 cells ( h ) with internalization inhibitors. Skin suspension cells were cultured with vehicle or MaR1 (10 nM) with nocodazole (10 μM), Pit stop2 (20 μM), nystatin (50 μg/mL), and chlorprozine (25 μg/mL) for 1 h and IL-23R expression was measured by flow cytometry. Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P

    Techniques Used: In Vitro, Cell Culture, Recombinant, Flow Cytometry, Cytometry, Fluorescence, Expressing

    2) Product Images from "Maresin-1 suppresses imiquimod-induced skin inflammation by regulating IL-23 receptor expression"

    Article Title: Maresin-1 suppresses imiquimod-induced skin inflammation by regulating IL-23 receptor expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-23623-9

    MaR1 inhibits IL-23 injection-induced psoriatic skin inflammation. ( a ) Ear swelling response. Ears swelling was measured from Day 0–16 upon topical application of vehicle or MaR1 (100 ng) 30 min before intradermal injection of 500 ng recombinant mouse IL-23 (n = 3) every other day. The mean ear thickness in both ears was calculated in 3 mice. ( b ) Hematoxylin and eosin-stained sections. Ear skin was collected on day 16. Bar, 200 μm. ( c ) Histological examination of epidermal thickness (n = 3). ( d ) The number of γδTCR mid+ and intracellular IL-17A + cells in the skin (n = 4). Ear skin was collected 24 h after IL-23 injection for two consecutive days and subjected to flow cytometry. ( e ) The number of CD4 + and intracellular IL-17A + cells in the skin (n = 4). Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P
    Figure Legend Snippet: MaR1 inhibits IL-23 injection-induced psoriatic skin inflammation. ( a ) Ear swelling response. Ears swelling was measured from Day 0–16 upon topical application of vehicle or MaR1 (100 ng) 30 min before intradermal injection of 500 ng recombinant mouse IL-23 (n = 3) every other day. The mean ear thickness in both ears was calculated in 3 mice. ( b ) Hematoxylin and eosin-stained sections. Ear skin was collected on day 16. Bar, 200 μm. ( c ) Histological examination of epidermal thickness (n = 3). ( d ) The number of γδTCR mid+ and intracellular IL-17A + cells in the skin (n = 4). Ear skin was collected 24 h after IL-23 injection for two consecutive days and subjected to flow cytometry. ( e ) The number of CD4 + and intracellular IL-17A + cells in the skin (n = 4). Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P

    Techniques Used: Injection, Recombinant, Mouse Assay, Staining, Flow Cytometry, Cytometry

    MaR1 treatment downregulates and internalizes IL-23 receptors in an in vitro skin assay. The frequencies of γδTCR mid+ and intracellular IL-17A + cells in IL-23 stimulated skin cell suspensions (n = 4). Skin cell suspensions were cultured with recombinant IL-23 with vehicle or MaR1 (10 nM) stimulation for 24 h, and the number of γδTCR mid+ and intracellular IL-17A + cells were analyzed by flow cytometry. ( b ) The number of CD4 + and intracellular IL-17A + cells in in IL-23 stimulated skin cell suspensions (n = 4). ( c , d ) The mean fluorescence intensity (MFI) of IL-23R expression on γδTCR mid+ ( c ) and CD4 + cells ( d ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 24 h and IL-23R expression was measured by flow cytometry. ( e , f ) The MFI of RORγt expression on γδTCR mid+ ( e ) and CD4 + cells ( f ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 3 h and intracellular RORγt expression was measured by flow cytometry. ( g , h ) The MFI of IL-23R expression on γδTCR mid+ ( g ) and CD4 cells ( h ) with internalization inhibitors. Skin suspension cells were cultured with vehicle or MaR1 (10 nM) with nocodazole (10 μM), Pit stop2 (20 μM), nystatin (50 μg/mL), and chlorprozine (25 μg/mL) for 1 h and IL-23R expression was measured by flow cytometry. Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P
    Figure Legend Snippet: MaR1 treatment downregulates and internalizes IL-23 receptors in an in vitro skin assay. The frequencies of γδTCR mid+ and intracellular IL-17A + cells in IL-23 stimulated skin cell suspensions (n = 4). Skin cell suspensions were cultured with recombinant IL-23 with vehicle or MaR1 (10 nM) stimulation for 24 h, and the number of γδTCR mid+ and intracellular IL-17A + cells were analyzed by flow cytometry. ( b ) The number of CD4 + and intracellular IL-17A + cells in in IL-23 stimulated skin cell suspensions (n = 4). ( c , d ) The mean fluorescence intensity (MFI) of IL-23R expression on γδTCR mid+ ( c ) and CD4 + cells ( d ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 24 h and IL-23R expression was measured by flow cytometry. ( e , f ) The MFI of RORγt expression on γδTCR mid+ ( e ) and CD4 + cells ( f ). Skin cell suspensions were cultured with vehicle or MaR1 (10 nM) for 3 h and intracellular RORγt expression was measured by flow cytometry. ( g , h ) The MFI of IL-23R expression on γδTCR mid+ ( g ) and CD4 cells ( h ) with internalization inhibitors. Skin suspension cells were cultured with vehicle or MaR1 (10 nM) with nocodazole (10 μM), Pit stop2 (20 μM), nystatin (50 μg/mL), and chlorprozine (25 μg/mL) for 1 h and IL-23R expression was measured by flow cytometry. Results are expressed as the mean ± SEM. All p -values were obtained by Student’s t test: * P

    Techniques Used: In Vitro, Cell Culture, Recombinant, Flow Cytometry, Cytometry, Fluorescence, Expressing

    3) Product Images from "Early MyD88-Dependent Induction of Interleukin-17A Expression during Salmonella Colitis ▿ Colitis ▿ †"

    Article Title: Early MyD88-Dependent Induction of Interleukin-17A Expression during Salmonella Colitis ▿ Colitis ▿ †

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00018-11

    IL-23 or IL-1β synergizes in inducing Il17a expression. Relative Il17a (A) and Il22 (B) transcript levels determined by quantitative real-time PCR after stimulation of splenic T cells with IL-23 or IL-1β for 5 h. Bars represent geometric means ± standard error of data from three independent experiments. Brackets indicate the statistical significance of differences. *, P
    Figure Legend Snippet: IL-23 or IL-1β synergizes in inducing Il17a expression. Relative Il17a (A) and Il22 (B) transcript levels determined by quantitative real-time PCR after stimulation of splenic T cells with IL-23 or IL-1β for 5 h. Bars represent geometric means ± standard error of data from three independent experiments. Brackets indicate the statistical significance of differences. *, P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    4) Product Images from "Identification of Canonical Tyrosine-dependent and Non-canonical Tyrosine-independent STAT3 Activation Sites in the Intracellular Domain of the Interleukin 23 Receptor *"

    Article Title: Identification of Canonical Tyrosine-dependent and Non-canonical Tyrosine-independent STAT3 Activation Sites in the Intracellular Domain of the Interleukin 23 Receptor *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.432153

    The cytoplasmic domains of mouse and human IL-23R induce STAT3 activation. A , the mouse IL-23 receptor complex consists of IL-12Rβ1, containing one tyrosine residue, box 1 and box 2 motifs, and the unique IL-23R with seven tyrosine residues. Six
    Figure Legend Snippet: The cytoplasmic domains of mouse and human IL-23R induce STAT3 activation. A , the mouse IL-23 receptor complex consists of IL-12Rβ1, containing one tyrosine residue, box 1 and box 2 motifs, and the unique IL-23R with seven tyrosine residues. Six

    Techniques Used: Activation Assay

    Differences in IL-23-mediated STAT3 activation in HeLa and Ba/F3-gp130 cells. A , IL-23R variants containing two or three mutations were generated by site-directed mutagenesis to define STAT3 recruitment sites within the cytoplasmic domain. B , p409 expression
    Figure Legend Snippet: Differences in IL-23-mediated STAT3 activation in HeLa and Ba/F3-gp130 cells. A , IL-23R variants containing two or three mutations were generated by site-directed mutagenesis to define STAT3 recruitment sites within the cytoplasmic domain. B , p409 expression

    Techniques Used: Activation Assay, Generated, Mutagenesis, Expressing

    Kinetics of IL-23-induced STAT3 activation. A , Ba/F3-gp130 cells stably transduced with cDNAs for murine IL-23R and murine IL-12Rβ1 were washed three times with PBS and starved for 2 h in serum-free DMEM. 1 × 10 6 cells were stimulated
    Figure Legend Snippet: Kinetics of IL-23-induced STAT3 activation. A , Ba/F3-gp130 cells stably transduced with cDNAs for murine IL-23R and murine IL-12Rβ1 were washed three times with PBS and starved for 2 h in serum-free DMEM. 1 × 10 6 cells were stimulated

    Techniques Used: Activation Assay, Stable Transfection, Transduction

    IL-23-mediated proliferation of stably transduced Ba/F3-gp130 cells involves PI3K/Akt activation. The indicated Ba/F3 cell lines were washed three times with PBS, starved for 4 h in serum-free medium, and pretreated for 60 min with P6 (1 μ m ),
    Figure Legend Snippet: IL-23-mediated proliferation of stably transduced Ba/F3-gp130 cells involves PI3K/Akt activation. The indicated Ba/F3 cell lines were washed three times with PBS, starved for 4 h in serum-free medium, and pretreated for 60 min with P6 (1 μ m ),

    Techniques Used: Stable Transfection, Activation Assay

    5) Product Images from "T-bet and Eomesodermin Play Critical Roles in Directing T Cell Differentiation to Th1 versus Th17"

    Article Title: T-bet and Eomesodermin Play Critical Roles in Directing T Cell Differentiation to Th1 versus Th17

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    IL-23 expands IL-6-initiated Th17 differentiation. A , Flow cytometry of intracellular IL-17 and IFN- γ expression in Stat6/T-bet DKO CD4 + CD25 – T cells stimulated with anti-CD3 (1 μ g/ml), IL-2 (1 ng/ml), and T-depleted wild-type
    Figure Legend Snippet: IL-23 expands IL-6-initiated Th17 differentiation. A , Flow cytometry of intracellular IL-17 and IFN- γ expression in Stat6/T-bet DKO CD4 + CD25 – T cells stimulated with anti-CD3 (1 μ g/ml), IL-2 (1 ng/ml), and T-depleted wild-type

    Techniques Used: Flow Cytometry, Cytometry, Expressing

    6) Product Images from "Intestinal macrophages arising from CCR2+ monocytes control pathogen infection by activating innate lymphoid cells"

    Article Title: Intestinal macrophages arising from CCR2+ monocytes control pathogen infection by activating innate lymphoid cells

    Journal: Nature Communications

    doi: 10.1038/ncomms9010

    Monocyte-derived intestinal macrophages are major producers of IL-1β and IL-23. ( a ) MP1 and DC1 subsets were isolated from uninfected (uninf) and C. rodentium -infected (inf) CD115 GFP animals. Cytokine mRNA expression was analysed by qPCR. Data are given as mean±s.d. ( n =5–7). * P
    Figure Legend Snippet: Monocyte-derived intestinal macrophages are major producers of IL-1β and IL-23. ( a ) MP1 and DC1 subsets were isolated from uninfected (uninf) and C. rodentium -infected (inf) CD115 GFP animals. Cytokine mRNA expression was analysed by qPCR. Data are given as mean±s.d. ( n =5–7). * P

    Techniques Used: Derivative Assay, Isolation, Infection, Expressing, Real-time Polymerase Chain Reaction

    Activation of RORγt + ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium ( C. rod ) infection. ( a ) CD3 - RORγt + ILCs from uninfected RORγt GFP/+ reporter mice and MP1 cells from C. rodentium -infected CD115 GFP were isolated. ILCs and MP1 cells (1 × 10 6 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml −1 ), IL-1β (10 μg ml −1 ), or isotype controls were used to block cytokines. Data are given as mean±s.d. ( n =4). * P
    Figure Legend Snippet: Activation of RORγt + ILCs requires IL-1β produced by monocyte-derived intestinal macrophages during C. rodentium ( C. rod ) infection. ( a ) CD3 - RORγt + ILCs from uninfected RORγt GFP/+ reporter mice and MP1 cells from C. rodentium -infected CD115 GFP were isolated. ILCs and MP1 cells (1 × 10 6 cells per ml) were cultured alone or co-cultured with or without stimulation with heat-killed C. rodentium (MOI=10) for 24 h. Neutralizing antibodies for IL-23 (10 μg ml −1 ), IL-1β (10 μg ml −1 ), or isotype controls were used to block cytokines. Data are given as mean±s.d. ( n =4). * P

    Techniques Used: Activation Assay, Produced, Derivative Assay, Infection, Mouse Assay, Isolation, Cell Culture, Blocking Assay

    7) Product Images from "Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokine-induced keratinocytes and skin"

    Article Title: Anti-inflammatory activity of compounds isolated from Astragalus sinicus L. in cytokine-induced keratinocytes and skin

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2013.157

    Amelioration of cytokine-induced cutaneous inflammation in mice skin by rf3 and rf4. ( a ) Mouse ears were topically treated with rf3 or rf4 and subsequently injected with phosphate-buffered saline or mouse IL-23 (500 ng/10 μl, each) into mouse ears every other day for 14 days ( n =5 mice per group). Histological assessment of the sections from the ears was performed by H E staining. ( b ) The mRNA levels of pro-inflammatory mediators were determined by quantitative real-time PCR in IL-23-injected mouse ears. Data were normalized to GAPDH signal and are presented as -fold change relative to the phosphate-buffered saline-treated group. Results are expressed as the mean±s.e.m. from three independent experiments. # P
    Figure Legend Snippet: Amelioration of cytokine-induced cutaneous inflammation in mice skin by rf3 and rf4. ( a ) Mouse ears were topically treated with rf3 or rf4 and subsequently injected with phosphate-buffered saline or mouse IL-23 (500 ng/10 μl, each) into mouse ears every other day for 14 days ( n =5 mice per group). Histological assessment of the sections from the ears was performed by H E staining. ( b ) The mRNA levels of pro-inflammatory mediators were determined by quantitative real-time PCR in IL-23-injected mouse ears. Data were normalized to GAPDH signal and are presented as -fold change relative to the phosphate-buffered saline-treated group. Results are expressed as the mean±s.e.m. from three independent experiments. # P

    Techniques Used: Mouse Assay, Injection, Staining, Real-time Polymerase Chain Reaction

    8) Product Images from "Sensitivity and resistance to regulation by IL-4 during Th17 maturation"

    Article Title: Sensitivity and resistance to regulation by IL-4 during Th17 maturation

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1002860

    TGFβ, IL-6 and IL-23 synergistically up-regulate IL-17 production during collagen re-stimulation, but IL-4 and IFNγ continue to suppress
    Figure Legend Snippet: TGFβ, IL-6 and IL-23 synergistically up-regulate IL-17 production during collagen re-stimulation, but IL-4 and IFNγ continue to suppress

    Techniques Used:

    9) Product Images from "Upregulation of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and suppression of Th17-mediated neutrophil recruitment in exogenous SOCS3 transfer in vitro"

    Article Title: Upregulation of SOCS3 in lung CD4+ T cells in a mouse model of chronic PA lung infection and suppression of Th17-mediated neutrophil recruitment in exogenous SOCS3 transfer in vitro

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.6630

    Decreased expression of p-STAT3 and RORγt in Pseudomonas aeruginosa-infected lung CD4+ T cells when stimulated by IL-23 following 72 h and 96 h of lentivirus-mediated SOCS3 gene transfection in vitro. (A) The protein expression of SOCS3, p-STAT3 and RORγt was determined by western blotting analysis. Histone H3 was used as an internal control. Protein quantification was performed by densitometry analysis using Quantity One software. (B) The mRNA level of SOCS3, STAT3 and RORγt was determined by reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as mean ± standard deviation of three separate experiments. *P
    Figure Legend Snippet: Decreased expression of p-STAT3 and RORγt in Pseudomonas aeruginosa-infected lung CD4+ T cells when stimulated by IL-23 following 72 h and 96 h of lentivirus-mediated SOCS3 gene transfection in vitro. (A) The protein expression of SOCS3, p-STAT3 and RORγt was determined by western blotting analysis. Histone H3 was used as an internal control. Protein quantification was performed by densitometry analysis using Quantity One software. (B) The mRNA level of SOCS3, STAT3 and RORγt was determined by reverse transcription-quantitative polymerase chain reaction analysis. Data are presented as mean ± standard deviation of three separate experiments. *P

    Techniques Used: Expressing, Infection, Transfection, In Vitro, Western Blot, Software, Real-time Polymerase Chain Reaction, Standard Deviation

    Suppressed Th17 responses in Pseudomonas aeruginosa-infected lung CD4+ T cells when stimulated by IL-23 following 72 and 96 h of lentivirus-mediated SOCS3 gene transfection. (A) Representative histograms of IL-17A+ cells by flow cytometry analysis. (B) The percentage of IL-17A+ cells in lung CD4+ T cells by flow cytometry analysis. (C) IL-17A level in the cell supernatant by ELISA. Data are presented as mean ± standard deviation of three separate experiments. *P
    Figure Legend Snippet: Suppressed Th17 responses in Pseudomonas aeruginosa-infected lung CD4+ T cells when stimulated by IL-23 following 72 and 96 h of lentivirus-mediated SOCS3 gene transfection. (A) Representative histograms of IL-17A+ cells by flow cytometry analysis. (B) The percentage of IL-17A+ cells in lung CD4+ T cells by flow cytometry analysis. (C) IL-17A level in the cell supernatant by ELISA. Data are presented as mean ± standard deviation of three separate experiments. *P

    Techniques Used: Infection, Transfection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

    10) Product Images from "Bacillus anthracis lethal toxin negatively modulates ILC3 function through perturbation of IL-23-mediated MAPK signaling"

    Article Title: Bacillus anthracis lethal toxin negatively modulates ILC3 function through perturbation of IL-23-mediated MAPK signaling

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006690

    In vivo administration of lethal toxin decreased IL-22 and GM-CSF production in ILC3s. Rag1 -/- mice were administered control solution or 100 μg of lethal toxin by tail vein injection. Two days post-injection, mice were euthanized and ( A ) the percentage and total number of ILC3s in livers, lungs and spleens from control mice (open circles) or lethal toxin treated mice (closed squares) were determined by flow cytometry. ( B-C ) Isolated lymphocytes were unstimulated (open circles) or stimulated with IL-23, PMA and ionomycin (black squares) in the presence of brefeldin A for 5 hrs. IL-22 and GM-CSF production was determined by intracellular flow cytometry. Shown is a representative experiment of 2 experiments with 3 mice per group. * p≤0.05, ** p≤0.01, *** p
    Figure Legend Snippet: In vivo administration of lethal toxin decreased IL-22 and GM-CSF production in ILC3s. Rag1 -/- mice were administered control solution or 100 μg of lethal toxin by tail vein injection. Two days post-injection, mice were euthanized and ( A ) the percentage and total number of ILC3s in livers, lungs and spleens from control mice (open circles) or lethal toxin treated mice (closed squares) were determined by flow cytometry. ( B-C ) Isolated lymphocytes were unstimulated (open circles) or stimulated with IL-23, PMA and ionomycin (black squares) in the presence of brefeldin A for 5 hrs. IL-22 and GM-CSF production was determined by intracellular flow cytometry. Shown is a representative experiment of 2 experiments with 3 mice per group. * p≤0.05, ** p≤0.01, *** p

    Techniques Used: In Vivo, Mouse Assay, Injection, Flow Cytometry, Cytometry, Isolation

    Destabilization of MEKs by lethal toxin in ILC3s. MNK-3 cells were pretreated with lethal toxin for 2 hr followed by IL-23 stimulation for 5–6 hr. ( A ) Il22 expression was analyzed by real time RT-PCR (n = 5), ( B) secreted IL-22 in supernatants was quantitated by ELISA (n = 4) and ( C ) the percent cells producing IL-22 (%IL-22 + ) was determined by intracellular cytokine staining (n = 3). * p≤0.05, ** p≤0.01, *** p
    Figure Legend Snippet: Destabilization of MEKs by lethal toxin in ILC3s. MNK-3 cells were pretreated with lethal toxin for 2 hr followed by IL-23 stimulation for 5–6 hr. ( A ) Il22 expression was analyzed by real time RT-PCR (n = 5), ( B) secreted IL-22 in supernatants was quantitated by ELISA (n = 4) and ( C ) the percent cells producing IL-22 (%IL-22 + ) was determined by intracellular cytokine staining (n = 3). * p≤0.05, ** p≤0.01, *** p

    Techniques Used: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

    Down-regulation of ERK1/2 and p38 activation in ILC3s by lethal toxin. MNK-3 cells were pretreated with lethal toxin (1 μg/ml) for 2 hr followed by IL-23 stimulation as indicated. Cell lysates were harvested and subjected to immunoblot analysis for MEK1, MEK2, MKK3, MKK6, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phosphorylated p38 (p-p38), total p38 (t-p38) and actin. Shown is a representative blot for n = 2–4 experiments. (B) MNK-3 cells were pretreated with anti-IL-23 p19 antibody (5 or 50 μg/ml) for 1 hr followed by IL-23 stimulation for 15 or 30 min. Cell lysates were subjected to immunoblot analysis for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated p38 (p-p38), total p38 and actin. Shown is a representative blot for 2–4 experiments. ( C ) In vitro expanded human Lin - CD127 + cells or ( D ) mouse Lin - CD127 + c-Kit + Thy1.2 + cells expanded from splenocytes of Rag1 -/- mice were treated with lethal toxin for 2 hr followed by IL-23 stimulation for 30 mins. Cell lysates were analyzed for MEK1, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and actin by immunoblot analysis. Shown are representative blots from 2 independent experiments for both C and D.
    Figure Legend Snippet: Down-regulation of ERK1/2 and p38 activation in ILC3s by lethal toxin. MNK-3 cells were pretreated with lethal toxin (1 μg/ml) for 2 hr followed by IL-23 stimulation as indicated. Cell lysates were harvested and subjected to immunoblot analysis for MEK1, MEK2, MKK3, MKK6, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 (t-ERK1/2), phosphorylated p38 (p-p38), total p38 (t-p38) and actin. Shown is a representative blot for n = 2–4 experiments. (B) MNK-3 cells were pretreated with anti-IL-23 p19 antibody (5 or 50 μg/ml) for 1 hr followed by IL-23 stimulation for 15 or 30 min. Cell lysates were subjected to immunoblot analysis for phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated p38 (p-p38), total p38 and actin. Shown is a representative blot for 2–4 experiments. ( C ) In vitro expanded human Lin - CD127 + cells or ( D ) mouse Lin - CD127 + c-Kit + Thy1.2 + cells expanded from splenocytes of Rag1 -/- mice were treated with lethal toxin for 2 hr followed by IL-23 stimulation for 30 mins. Cell lysates were analyzed for MEK1, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2 and actin by immunoblot analysis. Shown are representative blots from 2 independent experiments for both C and D.

    Techniques Used: Activation Assay, In Vitro, Mouse Assay

    Lethal toxin decreases IL-22 production in human ILCs. ( A ) Human tonsillar lymphocytes or ( B ) sorted Lin - CD127 + cells were pretreated with lethal toxin (1 μg/ml) for 2–3 hr followed by IL-23 (50 ng/ml) stimulation for 18 hrs. Cell supernatants were analyzed for IL-22 by ELISA. Shown is mean±SD of at least 10 donors (A) or 5 donors (B). * p≤0.05, ** p≤0.01, *** p
    Figure Legend Snippet: Lethal toxin decreases IL-22 production in human ILCs. ( A ) Human tonsillar lymphocytes or ( B ) sorted Lin - CD127 + cells were pretreated with lethal toxin (1 μg/ml) for 2–3 hr followed by IL-23 (50 ng/ml) stimulation for 18 hrs. Cell supernatants were analyzed for IL-22 by ELISA. Shown is mean±SD of at least 10 donors (A) or 5 donors (B). * p≤0.05, ** p≤0.01, *** p

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Inhibition of p38 signaling down-regulates IL-23-mediated IL-22 production in ILC3s. Rag1 -/- splenocytes were pretreated with DMSO or indicated concentration of ( A ) PD98059 (MEK1 inhibitor) or ( B ) SB203580 (p38 MAPK inhibitor) for 1 hr followed by IL-23 stimulation for 18 hr. IL-22 secretion was examined by ELISA. (C) Sorted Lin - CD127 + cells from Rag1 -/- splenocytes were pretreated with DMSO or indicated concentrations of PD98059 or SB203580 as in A and analyzed for IL-22 secretion by ELISA. In panel C shown is mean±SD from 3 experiments. * p≤0.05, ** p≤0.01, *** p
    Figure Legend Snippet: Inhibition of p38 signaling down-regulates IL-23-mediated IL-22 production in ILC3s. Rag1 -/- splenocytes were pretreated with DMSO or indicated concentration of ( A ) PD98059 (MEK1 inhibitor) or ( B ) SB203580 (p38 MAPK inhibitor) for 1 hr followed by IL-23 stimulation for 18 hr. IL-22 secretion was examined by ELISA. (C) Sorted Lin - CD127 + cells from Rag1 -/- splenocytes were pretreated with DMSO or indicated concentrations of PD98059 or SB203580 as in A and analyzed for IL-22 secretion by ELISA. In panel C shown is mean±SD from 3 experiments. * p≤0.05, ** p≤0.01, *** p

    Techniques Used: Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Lethal toxin down-regulates mouse IL-23-mediated production of IL-22 in ILC3s. ( A-C ) Rag1 -/- splenocytes were pretreated with (A) either lethal toxin (LeTx, lethal factor + protective antigen) or lethal factor (LF) (1 μg/ml) or (B) increasing doses of lethal toxin or (C) with enzymatic mutant toxin (E687C) for 3 hr followed by IL-23 (50 ng/ml) stimulation for 18 hr. Cell supernatants were analyzed for IL-22 secretion by ELISA. ( D-F ) Rag1 -/- splenocytes were treated with lethal toxin followed by IL-23 stimulation for 6 hr. Cell death was assessed by Annexin V and 7-AAD staining. (D) Shown are representative plots from one experiment of three. (E) Quantified apoptosis data and (F) IL-22 secretion from the same experiment are shown. ( G-H ) CD127 + ILCs were purified from spleens of Rag1 -/- mice and subjected to lethal toxin treatment as in A and measured for (G) Il22 mRNA expression and (H) IL-22 secretion after 6 hr of IL-23 stimulation. Shown are mean±SD from one experiment of at least three experiments (A-F). Shown are mean±SD from five experiments (G-H). * p≤0.05, ** p≤0.01, *** p
    Figure Legend Snippet: Lethal toxin down-regulates mouse IL-23-mediated production of IL-22 in ILC3s. ( A-C ) Rag1 -/- splenocytes were pretreated with (A) either lethal toxin (LeTx, lethal factor + protective antigen) or lethal factor (LF) (1 μg/ml) or (B) increasing doses of lethal toxin or (C) with enzymatic mutant toxin (E687C) for 3 hr followed by IL-23 (50 ng/ml) stimulation for 18 hr. Cell supernatants were analyzed for IL-22 secretion by ELISA. ( D-F ) Rag1 -/- splenocytes were treated with lethal toxin followed by IL-23 stimulation for 6 hr. Cell death was assessed by Annexin V and 7-AAD staining. (D) Shown are representative plots from one experiment of three. (E) Quantified apoptosis data and (F) IL-22 secretion from the same experiment are shown. ( G-H ) CD127 + ILCs were purified from spleens of Rag1 -/- mice and subjected to lethal toxin treatment as in A and measured for (G) Il22 mRNA expression and (H) IL-22 secretion after 6 hr of IL-23 stimulation. Shown are mean±SD from one experiment of at least three experiments (A-F). Shown are mean±SD from five experiments (G-H). * p≤0.05, ** p≤0.01, *** p

    Techniques Used: Mutagenesis, Enzyme-linked Immunosorbent Assay, Staining, Purification, Mouse Assay, Expressing

    11) Product Images from "Withasteroid B from D. metel L. regulates immune responses by modulating the JAK/STAT pathway and the IL‐17+RORγt+/IL‐10+FoxP3+ ratio"

    Article Title: Withasteroid B from D. metel L. regulates immune responses by modulating the JAK/STAT pathway and the IL‐17+RORγt+/IL‐10+FoxP3+ ratio

    Journal: Clinical and Experimental Immunology

    doi: 10.1111/cei.12998

    B2 ameliorates cutaneous inflammation in a mouse model. (a,b) Mouse ears were treated topically with B2 and subsequently injected intradermally with phosphate‐buffered saline (PBS) as a vehicle or mouse interleukin (IL)‐23 every other day for 14 days ( n = 6 mice/group). Ear swelling was evaluated 24 h after the final injection. Histology of mouse ear sections was assessed by haematoxylin and eosin (H E) staining. Acanthosis (c) and dermal cellular infiltrates (d) of skin injected with PBS or B2 collected from mice injected with IL‐23. * P
    Figure Legend Snippet: B2 ameliorates cutaneous inflammation in a mouse model. (a,b) Mouse ears were treated topically with B2 and subsequently injected intradermally with phosphate‐buffered saline (PBS) as a vehicle or mouse interleukin (IL)‐23 every other day for 14 days ( n = 6 mice/group). Ear swelling was evaluated 24 h after the final injection. Histology of mouse ear sections was assessed by haematoxylin and eosin (H E) staining. Acanthosis (c) and dermal cellular infiltrates (d) of skin injected with PBS or B2 collected from mice injected with IL‐23. * P

    Techniques Used: Injection, Mouse Assay, Staining

    12) Product Images from "Interleukin-17-producing ??+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor-?"

    Article Title: Interleukin-17-producing ??+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor-?

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2009.03166.x

    Non-diabetic non-obese diabetic (NOD) mice have a higher percentage of γδ + T cells than diabetic mice. (a, b) The percentage of γδ T cells in (a) freshly isolated splenocytes and (b) interleukin (IL)-23-expanded splenocytes
    Figure Legend Snippet: Non-diabetic non-obese diabetic (NOD) mice have a higher percentage of γδ + T cells than diabetic mice. (a, b) The percentage of γδ T cells in (a) freshly isolated splenocytes and (b) interleukin (IL)-23-expanded splenocytes

    Techniques Used: Mouse Assay, Isolation

    Interleukin (IL)-17 expression in non-obese diabetic (NOD) mice is dominated by CD4 − CD8 − γδ + T cells. (a, b) Freshly isolated (a) or IL-23-expanded (b) splenocytes from female NOD mice were double-stained with fluorescein
    Figure Legend Snippet: Interleukin (IL)-17 expression in non-obese diabetic (NOD) mice is dominated by CD4 − CD8 − γδ + T cells. (a, b) Freshly isolated (a) or IL-23-expanded (b) splenocytes from female NOD mice were double-stained with fluorescein

    Techniques Used: Expressing, Mouse Assay, Isolation, Staining

    Interleukin (IL)-23-expanded diabetic splenocytes produced higher levels of IL-17, but induced comparable diabetes in non-obese diabetic–severe combined immunodeficiency (NOD-SCID) mice compared with non-treated diabetic splenocytes. (a, b) Diabetic
    Figure Legend Snippet: Interleukin (IL)-23-expanded diabetic splenocytes produced higher levels of IL-17, but induced comparable diabetes in non-obese diabetic–severe combined immunodeficiency (NOD-SCID) mice compared with non-treated diabetic splenocytes. (a, b) Diabetic

    Techniques Used: Produced, Mouse Assay

    13) Product Images from "Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation"

    Article Title: Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation

    Journal: eLife

    doi: 10.7554/eLife.21330

    Glutaminolysis promotes T H 17 over iTreg cell fate by inhibiting N-glycan branching. ( A,B ) Flow cytometry of purified splenic CD4 + T cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23) ( A ) or in the presence of TGFβ ( B ). Gated on CD4 + . n = 3. DOI: http://dx.doi.org/10.7554/eLife.21330.014
    Figure Legend Snippet: Glutaminolysis promotes T H 17 over iTreg cell fate by inhibiting N-glycan branching. ( A,B ) Flow cytometry of purified splenic CD4 + T cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23) ( A ) or in the presence of TGFβ ( B ). Gated on CD4 + . n = 3. DOI: http://dx.doi.org/10.7554/eLife.21330.014

    Techniques Used: Flow Cytometry, Cytometry, Purification

    N-glycan branching controls T H 17 versus iTreg cell fate. ( A–F ) Flow cytometry ( A,B,E,F ), real-time qPCR ( C ) and UDP-GlcNAc LC-MS/MS analysis ( D ) of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) for 3 days ( D ) or 4 days ( A,B,E,F ) or as indicated ( C ). ( A ) gated on CD4 + IL-17A - or CD4 + IL-17A + as indicated. ( B,E,F ), gated on CD4. ( B ) Lactose (50 mM) is an inhibitor of galectin binding to branched N-glycans. ( A,B,F ) Unpaired two tailed t -test ( B ) with Bonferroni corrections ( A,F ). *p
    Figure Legend Snippet: N-glycan branching controls T H 17 versus iTreg cell fate. ( A–F ) Flow cytometry ( A,B,E,F ), real-time qPCR ( C ) and UDP-GlcNAc LC-MS/MS analysis ( D ) of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) for 3 days ( D ) or 4 days ( A,B,E,F ) or as indicated ( C ). ( A ) gated on CD4 + IL-17A - or CD4 + IL-17A + as indicated. ( B,E,F ), gated on CD4. ( B ) Lactose (50 mM) is an inhibitor of galectin binding to branched N-glycans. ( A,B,F ) Unpaired two tailed t -test ( B ) with Bonferroni corrections ( A,F ). *p

    Techniques Used: Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Purification, Binding Assay, Two Tailed Test

    N-glycan branching controls T H 17 versus iTreg cell fate via IL-2Rα (CD25). ( A–D,F–H ) Flow cytometry of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. ( D ) Co-incubation with doxycycline in vitro. ( A–C,D,F–H ) gated on CD4 + . ( A,B ) Mice treated with doxycycline in vivo, Mgat1 f/f tetO-Cre + ROSA rtTA gated on L-PHA - cells. ( A ) Gated on CD4 + IL-17A - or CD4 + IL-17A + as indicated. ( B ) Endocytosis analyzed by flow cytometry for CD25 internalization. Internalized percentage (MFI (internalized) / MFI (total) × 100%), normalized to control. ( D ) ELISA analysis of supernatant from purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23) as indicated for 4 days. ( A,B,E ) Unpaired two-tailed t -test with Welch’s ( A ) and Bonferroni corrections ( E ). *p
    Figure Legend Snippet: N-glycan branching controls T H 17 versus iTreg cell fate via IL-2Rα (CD25). ( A–D,F–H ) Flow cytometry of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. ( D ) Co-incubation with doxycycline in vitro. ( A–C,D,F–H ) gated on CD4 + . ( A,B ) Mice treated with doxycycline in vivo, Mgat1 f/f tetO-Cre + ROSA rtTA gated on L-PHA - cells. ( A ) Gated on CD4 + IL-17A - or CD4 + IL-17A + as indicated. ( B ) Endocytosis analyzed by flow cytometry for CD25 internalization. Internalized percentage (MFI (internalized) / MFI (total) × 100%), normalized to control. ( D ) ELISA analysis of supernatant from purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23) as indicated for 4 days. ( A,B,E ) Unpaired two-tailed t -test with Welch’s ( A ) and Bonferroni corrections ( E ). *p

    Techniques Used: Flow Cytometry, Cytometry, Purification, Incubation, In Vitro, Mouse Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Glycolysis promotes T H 17 over iTreg cell fate by inhibiting N-glycan branching. ( A ) Flow cytometry analysis of purified splenic CD4 + T cells activated with anti-CD3+ anti-CD28 for 2 days with T H 17 cytokines and as indicated for 1 hr with 2-NBDG. ( B ) Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of purified splenic CD4 + T cells at rest or activated with anti-CD3+anti-CD28 for 2 days with/without T H 17 cytokines (TGFβ+IL-6+IL-23) as indicated. ( C–G ) Flow cytometry ( C,D,F,G ) and Western blot ( E ) analysis of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23). Gated on CD4 + T cells. *p
    Figure Legend Snippet: Glycolysis promotes T H 17 over iTreg cell fate by inhibiting N-glycan branching. ( A ) Flow cytometry analysis of purified splenic CD4 + T cells activated with anti-CD3+ anti-CD28 for 2 days with T H 17 cytokines and as indicated for 1 hr with 2-NBDG. ( B ) Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of purified splenic CD4 + T cells at rest or activated with anti-CD3+anti-CD28 for 2 days with/without T H 17 cytokines (TGFβ+IL-6+IL-23) as indicated. ( C–G ) Flow cytometry ( C,D,F,G ) and Western blot ( E ) analysis of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17-inducing conditions (TGFβ+IL-6+IL-23). Gated on CD4 + T cells. *p

    Techniques Used: Flow Cytometry, Cytometry, Purification, Western Blot

    N-glycan branching controls T H 17 versus iTreg cell fate. ( A–C ) Flow cytometry of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. Gated on CD4 + . ( C ) Unpaired two tailed with Bonferroni corrections. *p
    Figure Legend Snippet: N-glycan branching controls T H 17 versus iTreg cell fate. ( A–C ) Flow cytometry of purified mouse splenic CD4 + T-cells activated with anti-CD3+anti-CD28 under T H 17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. Gated on CD4 + . ( C ) Unpaired two tailed with Bonferroni corrections. *p

    Techniques Used: Flow Cytometry, Cytometry, Purification, Two Tailed Test

    14) Product Images from "Galectin-7 downregulation in lesional keratinocytes contributes to enhanced IL-17A signaling and skin pathology in psoriasis"

    Article Title: Galectin-7 downregulation in lesional keratinocytes contributes to enhanced IL-17A signaling and skin pathology in psoriasis

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI130740

    Galectin-7–deficient mice exhibit hyperproliferative keratinocytes and increased leukocyte infiltration at the intradermally IL-23–injected sites, as compared with their littermate controls. ( A ) H E staining of ear sections from WT or galectin-7–deficient (knockout; KO) mice injected intradermally with PBS or IL-23 every other day for 15 days. Scale bars: 50 μm. ( B ) Ear thickness of WT and galectin-7–deficient (KO) mice was measured before each intradermal injection, and measurements were taken at the center of the ears (WT/PBS, n = 5; WT/IL-23, n = 18; KO/PBS, n = 5; KO/IL-23, n = 19). For statistical analysis, ear thickness of KO/IL-23 was compared with that in the corresponding WT/IL-23 group at each time point. ( C ) Epidermal thicknesses of WT and KO mice from H E-stained sections as described in A obtained on day 15 from the same mice as described in B . For each tissue section, 3 measurements were taken. ( D ) Leukocytes were counted in ×400 magnified visual fields of tissue sections from IL-23–injected mice using the cell counting module in ImageJ software (WT, n = 7; KO, n = 5). All results are presented as mean ± SD. For statistical analysis, 2-way ANOVA with Tukey’s multiple-comparison test ( B and C ) or unpaired Student’s t test ( D ) was performed. ** P
    Figure Legend Snippet: Galectin-7–deficient mice exhibit hyperproliferative keratinocytes and increased leukocyte infiltration at the intradermally IL-23–injected sites, as compared with their littermate controls. ( A ) H E staining of ear sections from WT or galectin-7–deficient (knockout; KO) mice injected intradermally with PBS or IL-23 every other day for 15 days. Scale bars: 50 μm. ( B ) Ear thickness of WT and galectin-7–deficient (KO) mice was measured before each intradermal injection, and measurements were taken at the center of the ears (WT/PBS, n = 5; WT/IL-23, n = 18; KO/PBS, n = 5; KO/IL-23, n = 19). For statistical analysis, ear thickness of KO/IL-23 was compared with that in the corresponding WT/IL-23 group at each time point. ( C ) Epidermal thicknesses of WT and KO mice from H E-stained sections as described in A obtained on day 15 from the same mice as described in B . For each tissue section, 3 measurements were taken. ( D ) Leukocytes were counted in ×400 magnified visual fields of tissue sections from IL-23–injected mice using the cell counting module in ImageJ software (WT, n = 7; KO, n = 5). All results are presented as mean ± SD. For statistical analysis, 2-way ANOVA with Tukey’s multiple-comparison test ( B and C ) or unpaired Student’s t test ( D ) was performed. ** P

    Techniques Used: Mouse Assay, Injection, Staining, Knock-Out, Cell Counting, Software

    The levels of galectin-7 are low in the epidermal keratinocytes of patients with psoriasis. ( A ) Representative immunohistochemical (IHC) staining pictures of galectin-7 in skin sections from a healthy control and a psoriatic lesion. Scale bars: 100 μm. ( B ) Quantification of IHC staining of galectin-7 in sections of normal skin from healthy controls ( n = 75) and sections of lesional skin from patients with psoriasis ( n = 27). Quantification of staining was performed by computer-assisted methods as described in Methods. The y axis indicates arbitrary numbers representing mean intensity across an area of the epidermis (InteDen/area). ( C ) IHC staining of galectin-7 in lesional skin and nonlesional skin pair. ( D ) Quantification of galectin-7 staining of paired lesional and nonlesional skin from the same patients ( n = 10 pairs). ( E ) IHC staining of galectin-7 in sections from intradermally IL-23–injected and PBS-injected mouse skin. Scale bars: 100 μm. ( F ) Quantification of galectin-7 staining of PBS-injected ( n = 6) and IL-23–injected mouse skin ( n = 13). ( G ) Immunoblot analysis of galectin-7 levels in HaCaT and HEKn cells stimulated with the indicated cytokines (IFN-γ, LPS, TNF-α, IL-23, or IL-17A). The concentrations of IFN-γ and LPS used were 100 ng/mL and 50 μg/mL, respectively. The concentrations of TNF-α, IL-23, and IL-17A used were as indicated in the figure. Cells were treated with the cytokines for 2 days, and cell lysates were prepared for immunoblot analysis. GAPDH served as a loading control. The intensity of the galectin-7 band was quantified by densitometry and normalized to GAPDH and control samples. The number below each band represents the relative galectin-7 amount. Statistical analyses were performed by unpaired ( B and F ) and paired ( D ) Student’s t test. * P
    Figure Legend Snippet: The levels of galectin-7 are low in the epidermal keratinocytes of patients with psoriasis. ( A ) Representative immunohistochemical (IHC) staining pictures of galectin-7 in skin sections from a healthy control and a psoriatic lesion. Scale bars: 100 μm. ( B ) Quantification of IHC staining of galectin-7 in sections of normal skin from healthy controls ( n = 75) and sections of lesional skin from patients with psoriasis ( n = 27). Quantification of staining was performed by computer-assisted methods as described in Methods. The y axis indicates arbitrary numbers representing mean intensity across an area of the epidermis (InteDen/area). ( C ) IHC staining of galectin-7 in lesional skin and nonlesional skin pair. ( D ) Quantification of galectin-7 staining of paired lesional and nonlesional skin from the same patients ( n = 10 pairs). ( E ) IHC staining of galectin-7 in sections from intradermally IL-23–injected and PBS-injected mouse skin. Scale bars: 100 μm. ( F ) Quantification of galectin-7 staining of PBS-injected ( n = 6) and IL-23–injected mouse skin ( n = 13). ( G ) Immunoblot analysis of galectin-7 levels in HaCaT and HEKn cells stimulated with the indicated cytokines (IFN-γ, LPS, TNF-α, IL-23, or IL-17A). The concentrations of IFN-γ and LPS used were 100 ng/mL and 50 μg/mL, respectively. The concentrations of TNF-α, IL-23, and IL-17A used were as indicated in the figure. Cells were treated with the cytokines for 2 days, and cell lysates were prepared for immunoblot analysis. GAPDH served as a loading control. The intensity of the galectin-7 band was quantified by densitometry and normalized to GAPDH and control samples. The number below each band represents the relative galectin-7 amount. Statistical analyses were performed by unpaired ( B and F ) and paired ( D ) Student’s t test. * P

    Techniques Used: Immunohistochemistry, Staining, Injection

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    Incubation:

    Article Title: Interleukin-17-producing ??+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor-?
    Article Snippet: Cytokine secretion by splenocytes or pancreatic draining lymph node cells was determined by ELISA as recommended by the kit manufacturer (eBioscience). .. Briefly, cells from female NOD mice (5 × 105 ) were incubated in 96-well flat-bottomed microtitre plates with 0·1 μg/ml of anti-CD3 antibody (1452C11) in the presence or absence of 10 ng/ml of recombinant mouse IL-23 (eBioscience) and the supernatants were harvested after 48 hr. .. Levels of IL-17, IFN-γ, TGF-β and IL-10 were determined in triplicate in 0·1 ml of supernatant using a sandwich ELISA method.

    Injection:

    Article Title: Macrophage Migration Inhibitory Factor (MIF) Drives Murine Psoriasiform Dermatitis
    Article Snippet: Induction of recombinant IL-23-induced dermatitis IL-23-induced dermatitis was induced and evaluated, as previously described ( ). .. Briefly, 0.5 μg of recombinant murine IL-23 (eBioscience, Frankfurt, Germany), solved in 1% BSA in PBS, was injected i.d. in a volume of 20 μl into the dorsal surface of right ear, vehicle control (1% BSA in PBS) into the left ear of recipient mice every other day, starting day 0 of the experiment. .. Generation of bone marrow chimera Bone marrow chimera were generated, as described previously ( ).

    Isolation:

    Article Title: Early MyD88-Dependent Induction of Interleukin-17A Expression during Salmonella Colitis ▿ Colitis ▿ †
    Article Snippet: Cells were washed and placed in complete IMDM containing 25 μg/ml of gentamicin for an additional 3 h. Isolation of untouched splenic T cells was performed using a pan-T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per the manufacturer's instructions. .. Cells were treated with 10 ng/ml of recombinant mouse IL-23 and/or 10 ng/ml of IL-1β (eBioscience, San Diego, CA), for a total of 5 h. RNA was isolated using a Qiagen Qiashredder/RNEasy kit per the manufacturer's instructions. .. Three groups of two naive mice each (C57BL/6; Taconic) were euthanized, and organ tissue samples from each group of mice were combined.

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    Thermo Fisher tnf α
    Indoleamine 2,3 dioxygenase-1 (IDO1) influences the gene expression of cytokines and transcription factors. Relative expression of mRNA for aryl hydrocarbon receptor (AhR), IFN-γ, <t>TNF-α,</t> IL-6, RORC, Tbet, GATA3, FoxP3, IL-10, TGF-β, IL-17, and IL-22 in whole lung cells of wild-type and IDO1 −/− mice after 10 weeks of Paracoccidioides brasiliensis infection. The level of gene transcription was determined by real-time PCR. Bars show mean ± SEM from at least four mice per group and are representative of three independent experiments (* p
    Tnf α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse il 23
    CX 3 CR1 + MNP-derived TL1A synergizes with <t>IL-23</t> and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.
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    Analysis of T-cell activation after immunization with the RNA adjuvant and MERS S protein vaccine. ( A ) The population of MERS S protein-specific cells secreting interferon-γ (IFN-γ) and <t>interleukin-2</t> (IL-2) were quantified by using ELISPOT assay, after treatment with MERS S protein in cultured splenocytes from immunized mice. ( B ) Cytokine levels in splenocyte supernatants stimulated with MERS S protein from immunized mice were measured, using ELISA. ( C ) IFN-γ, IL-2, and tumor necrosis factor α (TNF-α)-producing CD4+ T-cells in splenocytes were counted by flow cytometry. ( D ) IFN-γ, IL-2, and TNF-α producing polyfunctional CD4+ T-cells in splenocytes were analyzed by flow cytometry. *, p
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    IL-17 production in DC-T cell cocultures stimulated with schistosome eggs or rIL-6, TGF- β , and <t>IL-23.</t> A–F , Naive CD4 T cells from CBA and BL/6 mice were cocultured with syngeneic BMDC in the presence or absence of anti-CD3/anti-CD28 coated
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    Indoleamine 2,3 dioxygenase-1 (IDO1) influences the gene expression of cytokines and transcription factors. Relative expression of mRNA for aryl hydrocarbon receptor (AhR), IFN-γ, TNF-α, IL-6, RORC, Tbet, GATA3, FoxP3, IL-10, TGF-β, IL-17, and IL-22 in whole lung cells of wild-type and IDO1 −/− mice after 10 weeks of Paracoccidioides brasiliensis infection. The level of gene transcription was determined by real-time PCR. Bars show mean ± SEM from at least four mice per group and are representative of three independent experiments (* p

    Journal: Frontiers in Immunology

    Article Title: The IDO–AhR Axis Controls Th17/Treg Immunity in a Pulmonary Model of Fungal Infection

    doi: 10.3389/fimmu.2017.00880

    Figure Lengend Snippet: Indoleamine 2,3 dioxygenase-1 (IDO1) influences the gene expression of cytokines and transcription factors. Relative expression of mRNA for aryl hydrocarbon receptor (AhR), IFN-γ, TNF-α, IL-6, RORC, Tbet, GATA3, FoxP3, IL-10, TGF-β, IL-17, and IL-22 in whole lung cells of wild-type and IDO1 −/− mice after 10 weeks of Paracoccidioides brasiliensis infection. The level of gene transcription was determined by real-time PCR. Bars show mean ± SEM from at least four mice per group and are representative of three independent experiments (* p

    Article Snippet: The levels of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-22, IL-27, IL-35, IL-23, TNF-α, IFN-γ, and TGF-β were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from eBioscience or PBL.

    Techniques: Expressing, Mouse Assay, Infection, Real-time Polymerase Chain Reaction

    Indoleamine 2,3 dioxygenase-1 (IDO1) expression controls the presence of intracellular cytokines in pulmonary CD11b + and CD11c + cells. The intracellular cytokines were determined in CD11b + (A) and CD11c + (B) lung infiltrating leukocytes of wild-type and IDO1 −/− C57BL/6 mice after 96 h, 2, and 10 weeks of infection with 1 × 10 6 Paracoccidioides brasiliensis yeasts. The lung cells were obtained as described in Section “ Materials and Methods ” and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD11b + or CD11c + expression and then for the presence of cytokines IL-12, TNF-α, IL-1β, IL-10, IL-6, and TGF-β. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (* p

    Journal: Frontiers in Immunology

    Article Title: The IDO–AhR Axis Controls Th17/Treg Immunity in a Pulmonary Model of Fungal Infection

    doi: 10.3389/fimmu.2017.00880

    Figure Lengend Snippet: Indoleamine 2,3 dioxygenase-1 (IDO1) expression controls the presence of intracellular cytokines in pulmonary CD11b + and CD11c + cells. The intracellular cytokines were determined in CD11b + (A) and CD11c + (B) lung infiltrating leukocytes of wild-type and IDO1 −/− C57BL/6 mice after 96 h, 2, and 10 weeks of infection with 1 × 10 6 Paracoccidioides brasiliensis yeasts. The lung cells were obtained as described in Section “ Materials and Methods ” and labeled with antibodies conjugated to different fluorochromes. The lung infiltrating leukocytes were gated by FSC/SSC analysis. The cells were gated for CD11b + or CD11c + expression and then for the presence of cytokines IL-12, TNF-α, IL-1β, IL-10, IL-6, and TGF-β. One hundred thousand cells were acquired on FACS CANTO II and subsequently analyzed by FlowJo software. Data are expressed as means ± SEM and are representative of three independent experiments using five mice of each mouse strain per group (* p

    Article Snippet: The levels of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, IL-22, IL-27, IL-35, IL-23, TNF-α, IFN-γ, and TGF-β were measured by capture enzyme-linked immunosorbent assay (ELISA) with antibody pairs purchased from eBioscience or PBL.

    Techniques: Expressing, Mouse Assay, Infection, Labeling, FACS, Software

    CX 3 CR1 + MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: CX 3 CR1 + MNP-derived TL1A synergizes with IL-23 and IL-1β to induce IL-22 in intestinal ILC3. (A) Gene-set enrichment analysis was used to determine whether the indicated disease-related SNP were differentially expressed between CD14 + MNPs and CD103 + DCs. Significance was estimated using the hypergeometric cumulative distribution, with a raw p-value cutoff of 0.05 for differential expression. Data were averaged from two independent donors. (B) B cells (CD3 − CD19 + ), CX 3 CR1 + MNPs, CD103 + DCs, and Ly6C + monocytes were sorted from the intestinal lamina propria of Cx3cr1 GFP/+ and quantitative PCR for TL1A was performed. Relative quantitation was performed by ΔCt normalized to GAPDH expression. Data are from two biological replicates performed with two technical replicates. *, P ≤ 0.05. Two-tailed Student’s t test. (C–E) Sorted intestinal ILCs from mice (C and E) or cultured human intestinal ILCs (D) were stimulated with media alone, IL-1β, or IL-23 with or without TL1A as indicated for 18 h. Brefeldin was added to the cultures 4 h before intracellular cytokine staining for IL-22 (C and D) or GM-CSF (E). Data are representative of six independent experiments. (F–H) Sorted intestinal ILCs were transfected with siRNA targeting Tnfrsf25 or a scramble control. (F) Knockdown efficiency was assessed after 24 h by flow cytometry comparing scramble control (solid line) with Tnfrsf25 siRNA. One of two representative experiments is shown. ILCs were then cultured with media alone (-) or IL-23 and TL1A or co-cultured with CX 3 CR1 + MNPs with or without LPS as indicated for an additional 18 h. (G) IL-22 production was measured by intracellular flow cytometry. Brefeldin was added to the cultures 4 h before intracellular cytokine staining. Data are representative of two independent experiments. (H) IL-22 secretion by samples from G were assessed by ELISA, performed in duplicate, before addition of Brefeldin. *, P ≤ 0.05; **, P ≤ 0.01. Two-tailed Student’s t test. Error bars represent SEM.

    Article Snippet: Human and mouse ILCs were stimulated with human or mouse IL-23 (eBioscience; 40 ng/ml), IL-1β (eBioscience; 10 ng/ml), or TL1A (R & D Systems; 200 ng/ml) as indicated, respectively.

    Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Quantitation Assay, Two Tailed Test, Mouse Assay, Cell Culture, Staining, Transfection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease ( n = 8, gray) or ulcerative colitis ( n = 6, black; Table S1 ), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy ( n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3 + or CD3 − fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3 − IL-22 + (black lines) and CD3 − IL-22 − (gray) LPMCs. (C) Expression of RORγt by c-Kit + CD56 + LPMCs. Lin − cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ − ; Fig. S3 A ) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin − CD56 + c-Kit + ILC3 (black line) were compared with Lin − CD56 + c-Kit − NK cells (gray) for RORγt expression. (D) Surface staining of Lin − c-Kit + ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin − CD56 + c-Kit + . The isotype control is in gray. (F) CD11c + MHCII + human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22 + ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: Increased ILC3 production of IL-22 in mild to moderate IBD correlates with presence of fecal stream. (A) LPMCs isolated from descending colon biopsies from patients with endoscopically mild to moderate Crohn’s’ disease ( n = 8, gray) or ulcerative colitis ( n = 6, black; Table S1 ), as well as age-matched non-IBD control patients undergoing routine screening colonoscopy ( n = 8, white), were stimulated ex vivo with PMA/ionomycin and evaluated by intracellular cytokine staining for expression of IL-17 and IL-22. The percentage of CD3 + or CD3 − fraction expressing IL-17 or IL-22 is indicated. *, P ≤ 0.05, two-tailed Student’s t test. Black bars represent the geometric mean. (B) Expression of c-Kit and CD56 in electronically gated CD3 − IL-22 + (black lines) and CD3 − IL-22 − (gray) LPMCs. (C) Expression of RORγt by c-Kit + CD56 + LPMCs. Lin − cells (CD14/CD19/CD3/CD11b/CD11c/TCRγδ − ; Fig. S3 A ) were stained with antibodies to surface markers c-Kit and CD56 and to intracellular RORγt. Lin − CD56 + c-Kit + ILC3 (black line) were compared with Lin − CD56 + c-Kit − NK cells (gray) for RORγt expression. (D) Surface staining of Lin − c-Kit + ILCs for the indicated markers (black line) compared with isotype control (gray) and all live LPMCs (dotted line) (E) LPMCs stained for intracellular IL-22 after stimulation with IL-23 for 3 h (solid line) or with control media (dotted line). Cells shown were gated on Lin − CD56 + c-Kit + . The isotype control is in gray. (F) CD11c + MHCII + human colonic APCs were electronically gated for expression of CD103 and CD14. One of three donors is shown. (G) Lamina propria cells from biopsy samples of tissue exposed (prediversion) or not exposed to the fecal stream (post-diversion) were cultured for 3 h and ILC3 production of IL-22 was assessed by flow cytometry. (left) Result from one representative donor. (right) Percentage of IL-22 + ILCs in afferent (Pre) and efferent (Post) limbs of three diverted patients. **, P ≤ 0.01, two-tailed Student’s t test. Black bars represent the geometric mean.

    Article Snippet: Human and mouse ILCs were stimulated with human or mouse IL-23 (eBioscience; 40 ng/ml), IL-1β (eBioscience; 10 ng/ml), or TL1A (R & D Systems; 200 ng/ml) as indicated, respectively.

    Techniques: Isolation, Ex Vivo, Staining, Expressing, Two Tailed Test, Cell Culture, Flow Cytometry, Cytometry

    TLR-stimulated CX 3 CR1 + MNPs are stronger inducers of ILC3 production of IL-22 than CD103 + CD11b + DCs and monocytes. (A–C) CD103 or CX 3 CR1 + MHCII + CD11c + CD11b + cells were isolated from the lamina propria of CX 3 CR1 GFP/+ mice (sort strategy shown in A and co-cultured with Lin − RORγt-GFP + ILCs with or without the indicated bacterial TLR ligands or IL-23. IL-22 was assessed by intracellular staining of CD90.2 + ILCs after 18 h. A representative flow cytometry plot is shown in B. (C) Percent IL-22 + ILCs is shown from seven independent experiments. **, P ≤ 0.01; *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent SEM. (D–F) Ly6C + MHCII lo (monocytes) and Ly6C − MHCII hi (MNPs) were isolated from CX 3 CR1 + CD11b + lamina propria cells (sort strategy is shown in D and co-cultured with intestinal ILCs with LPS or IL-23 as indicated. Intracellular cytokine staining for IL-22 is shown after 18 h (E). Supernatants were harvested after 18 h and IL-22 production quantitated by ELISA (F). Results are representative of two independent experiments performed in triplicate. ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: TLR-stimulated CX 3 CR1 + MNPs are stronger inducers of ILC3 production of IL-22 than CD103 + CD11b + DCs and monocytes. (A–C) CD103 or CX 3 CR1 + MHCII + CD11c + CD11b + cells were isolated from the lamina propria of CX 3 CR1 GFP/+ mice (sort strategy shown in A and co-cultured with Lin − RORγt-GFP + ILCs with or without the indicated bacterial TLR ligands or IL-23. IL-22 was assessed by intracellular staining of CD90.2 + ILCs after 18 h. A representative flow cytometry plot is shown in B. (C) Percent IL-22 + ILCs is shown from seven independent experiments. **, P ≤ 0.01; *, P ≤ 0.05. One way ANOVA with Bonferroni’s correction. Error bars represent SEM. (D–F) Ly6C + MHCII lo (monocytes) and Ly6C − MHCII hi (MNPs) were isolated from CX 3 CR1 + CD11b + lamina propria cells (sort strategy is shown in D and co-cultured with intestinal ILCs with LPS or IL-23 as indicated. Intracellular cytokine staining for IL-22 is shown after 18 h (E). Supernatants were harvested after 18 h and IL-22 production quantitated by ELISA (F). Results are representative of two independent experiments performed in triplicate. ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Article Snippet: Human and mouse ILCs were stimulated with human or mouse IL-23 (eBioscience; 40 ng/ml), IL-1β (eBioscience; 10 ng/ml), or TL1A (R & D Systems; 200 ng/ml) as indicated, respectively.

    Techniques: Isolation, Mouse Assay, Cell Culture, Staining, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    CX 3 CR1 + MNP-derived IL-23 and IL-1β activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1 STOP-DTR/GFP mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX 3 CR1 hi MNPs. (bottom) Expression of Ly6C and MHCII on CX 3 CR1 hi and CX 3 CR1 int populations. (B) Expression of IL-22 in Lin − CD90.2 + colonic ILCs from Cx3cr1 STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1 STOP-DTR/+ (Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days −2, −1, and 0 and every other day postinfection. One representative intracellular cytokine flow cytometry plot is shown on the left and a composite graph ( n = 6/group) on the right. *, P ≤ 0.05, two-tailed Student’s t test. Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures ( Fig. 3, B and C ) were harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX 3 CR1 + MNPs or CD103 + CD11b + DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1β by ELISA. Results are the mean of two independent experiments performed in duplicate and the SEM is shown. *, P ≤ 0.05; **, P ≤ 0.01. (E) Lin − CD90.2 hi ILCs from WT or Il1r −/− mice were co-cultured with sorted intestinal MNPs from WT or Il23p19 −/− mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after 18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P ≤ 0.05; ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: CX 3 CR1 + MNP-derived IL-23 and IL-1β activate ILC3 to produce IL-22. (A) Phenotype analysis of colonic LPMCs from Cx3cr1 STOP-DTR/GFP mice with or without CD11c-cre after DT injection for 2 d. (top) Selective depletion of CX 3 CR1 hi MNPs. (bottom) Expression of Ly6C and MHCII on CX 3 CR1 hi and CX 3 CR1 int populations. (B) Expression of IL-22 in Lin − CD90.2 + colonic ILCs from Cx3cr1 STOP-DTR/+ (Stop-DTR) or CD11c-Cre x Cx3cr1 STOP-DTR/+ (Cre-DTR) mice at 7 d after C. rodentium infection. DT was administered at days −2, −1, and 0 and every other day postinfection. One representative intracellular cytokine flow cytometry plot is shown on the left and a composite graph ( n = 6/group) on the right. *, P ≤ 0.05, two-tailed Student’s t test. Error bars represent the SEM. Results are a composite of two independent experiments. (C) Supernatants from APC-ILC co-cultures ( Fig. 3, B and C ) were harvested after 18 h and assayed for IL-23 by ELISA. Results are averages of three independent experiments and the SEM is shown. (D) CX 3 CR1 + MNPs or CD103 + CD11b + DCs were sorted and incubated with media or CpG for 18 h and supernatants were assayed for IL-1β by ELISA. Results are the mean of two independent experiments performed in duplicate and the SEM is shown. *, P ≤ 0.05; **, P ≤ 0.01. (E) Lin − CD90.2 hi ILCs from WT or Il1r −/− mice were co-cultured with sorted intestinal MNPs from WT or Il23p19 −/− mice, with or without CpG, as indicated. IL-22 production by the ILCs was assessed after 18 h by ELISA. Data are combined from three independent experiments performed in duplicate. *, P ≤ 0.05; ***, P ≤ 0.001. One-way ANOVA with Bonferroni correction. Error bars represent the SEM.

    Article Snippet: Human and mouse ILCs were stimulated with human or mouse IL-23 (eBioscience; 40 ng/ml), IL-1β (eBioscience; 10 ng/ml), or TL1A (R & D Systems; 200 ng/ml) as indicated, respectively.

    Techniques: Derivative Assay, Mouse Assay, Injection, Expressing, Infection, Flow Cytometry, Cytometry, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Incubation, Cell Culture

    Human ILC3 production of IL-22 is supported by IL-23 and IL-1β produced by TLR-stimulated CD14 + and CX 3 CR1 + MNPs. (A–C) HLA-DR + CD11c + cells from intestinal resection tissue were sorted into CD103 + DCs and CD14 + MNPs subpopulations and transcriptional profiles were assessed by RNA-seq. (A) Sorting strategy. (B) Each subset was examined for expression of the indicated cell surface markers. Isotype controls are shown in gray. One of three donors is shown. (C) Heatmap of relative expression of relevant MNP-related genes. Values represent the mean of two independent donors, and an asterisk denotes individual genes differentially expressed at an FDR = 0.01. (D and E) Induction of IL-22 in human ILCs in co-culture with CD14 + MNPs or CD103 + DCs in the presence of media alone, LPS, or flagellin, as indicated. c-Kit + cells were examined for intracellular IL-22 production after 18-h culture. Data are representative of five independent experiments. (F) CD14 + MNPs or CD103 + DCs sorted from human intestine (as in A) were stimulated with the indicated TLR ligands for 18 h and qPCR or cytometric bead array analysis were used to quantitate IL-23p19 and IL-1β, respectively. Results are averaged from three independent donors, and technical replicates were performed in duplicate or triplicate, respectively. *, P ≤ 0.05. N.D., not detected. Two-tailed Student’s t test. Error bars represent the SEM. (G) Sorted human intestinal CD14 + MNPs and ILCs were left unstimulated or were co-cultured in the presence of LPS with or without neutralizing antibodies against IL-1β and IL-23. IL-22 ELISA was performed after 18h. Results are averaged from two independent donors performed in duplicate. *, P ≤ 0.05. Two-tailed Student’s t test. Error bars represent the SEM.

    Journal: The Journal of Experimental Medicine

    Article Title: CX3CR1+ mononuclear phagocytes support colitis-associated innate lymphoid cell production of IL-22

    doi: 10.1084/jem.20140678

    Figure Lengend Snippet: Human ILC3 production of IL-22 is supported by IL-23 and IL-1β produced by TLR-stimulated CD14 + and CX 3 CR1 + MNPs. (A–C) HLA-DR + CD11c + cells from intestinal resection tissue were sorted into CD103 + DCs and CD14 + MNPs subpopulations and transcriptional profiles were assessed by RNA-seq. (A) Sorting strategy. (B) Each subset was examined for expression of the indicated cell surface markers. Isotype controls are shown in gray. One of three donors is shown. (C) Heatmap of relative expression of relevant MNP-related genes. Values represent the mean of two independent donors, and an asterisk denotes individual genes differentially expressed at an FDR = 0.01. (D and E) Induction of IL-22 in human ILCs in co-culture with CD14 + MNPs or CD103 + DCs in the presence of media alone, LPS, or flagellin, as indicated. c-Kit + cells were examined for intracellular IL-22 production after 18-h culture. Data are representative of five independent experiments. (F) CD14 + MNPs or CD103 + DCs sorted from human intestine (as in A) were stimulated with the indicated TLR ligands for 18 h and qPCR or cytometric bead array analysis were used to quantitate IL-23p19 and IL-1β, respectively. Results are averaged from three independent donors, and technical replicates were performed in duplicate or triplicate, respectively. *, P ≤ 0.05. N.D., not detected. Two-tailed Student’s t test. Error bars represent the SEM. (G) Sorted human intestinal CD14 + MNPs and ILCs were left unstimulated or were co-cultured in the presence of LPS with or without neutralizing antibodies against IL-1β and IL-23. IL-22 ELISA was performed after 18h. Results are averaged from two independent donors performed in duplicate. *, P ≤ 0.05. Two-tailed Student’s t test. Error bars represent the SEM.

    Article Snippet: Human and mouse ILCs were stimulated with human or mouse IL-23 (eBioscience; 40 ng/ml), IL-1β (eBioscience; 10 ng/ml), or TL1A (R & D Systems; 200 ng/ml) as indicated, respectively.

    Techniques: Produced, RNA Sequencing Assay, Expressing, Co-Culture Assay, Real-time Polymerase Chain Reaction, Two Tailed Test, Cell Culture, Enzyme-linked Immunosorbent Assay

    Analysis of T-cell activation after immunization with the RNA adjuvant and MERS S protein vaccine. ( A ) The population of MERS S protein-specific cells secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) were quantified by using ELISPOT assay, after treatment with MERS S protein in cultured splenocytes from immunized mice. ( B ) Cytokine levels in splenocyte supernatants stimulated with MERS S protein from immunized mice were measured, using ELISA. ( C ) IFN-γ, IL-2, and tumor necrosis factor α (TNF-α)-producing CD4+ T-cells in splenocytes were counted by flow cytometry. ( D ) IFN-γ, IL-2, and TNF-α producing polyfunctional CD4+ T-cells in splenocytes were analyzed by flow cytometry. *, p

    Journal: Pharmaceutics

    Article Title: MERS-CoV Spike Protein Vaccine and Inactivated Influenza Vaccine Formulated with Single Strand RNA Adjuvant Induce T-Cell Activation through Intranasal Immunization in Mice

    doi: 10.3390/pharmaceutics12050441

    Figure Lengend Snippet: Analysis of T-cell activation after immunization with the RNA adjuvant and MERS S protein vaccine. ( A ) The population of MERS S protein-specific cells secreting interferon-γ (IFN-γ) and interleukin-2 (IL-2) were quantified by using ELISPOT assay, after treatment with MERS S protein in cultured splenocytes from immunized mice. ( B ) Cytokine levels in splenocyte supernatants stimulated with MERS S protein from immunized mice were measured, using ELISA. ( C ) IFN-γ, IL-2, and tumor necrosis factor α (TNF-α)-producing CD4+ T-cells in splenocytes were counted by flow cytometry. ( D ) IFN-γ, IL-2, and TNF-α producing polyfunctional CD4+ T-cells in splenocytes were analyzed by flow cytometry. *, p

    Article Snippet: The concentrations of interferon γ (IFN-γ), interleukin-2 (IL-2), IL-6, and tumor necrosis factor α (TNF-α) were detected with ELISA kits (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions.

    Techniques: Activation Assay, Enzyme-linked Immunospot, Cell Culture, Mouse Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    IL-17 production in DC-T cell cocultures stimulated with schistosome eggs or rIL-6, TGF- β , and IL-23. A–F , Naive CD4 T cells from CBA and BL/6 mice were cocultured with syngeneic BMDC in the presence or absence of anti-CD3/anti-CD28 coated

    Journal:

    Article Title: Dendritic Cell IL-23 and IL-1 Production in Response to Schistosome Eggs Induces Th17 Cells in a Mouse Strain Prone to Severe Immunopathology 1

    doi:

    Figure Lengend Snippet: IL-17 production in DC-T cell cocultures stimulated with schistosome eggs or rIL-6, TGF- β , and IL-23. A–F , Naive CD4 T cells from CBA and BL/6 mice were cocultured with syngeneic BMDC in the presence or absence of anti-CD3/anti-CD28 coated

    Article Snippet: After 4 days, the culture supernatants were removed and assayed by ELISA for IL-17, IL-6, and IFN- γ using Ab, standards, and protocols from R & D Systems; for IL-23, using Ab, standards, and protocols from eBioscience; and for IL-5, using Ab, standards, and protocols from BD Pharmingen.

    Techniques: Crocin Bleaching Assay, Mouse Assay

    Neutralization of IL-23 and IL-1 inhibits egg-induced IL-17 production in CBA DC-T cell cocultures. A and B , DC-T cell cocultures were established in the presence or absence of eggs, anti-CD3/anti-CD28 coated beads, and the indicated blocking reagents

    Journal:

    Article Title: Dendritic Cell IL-23 and IL-1 Production in Response to Schistosome Eggs Induces Th17 Cells in a Mouse Strain Prone to Severe Immunopathology 1

    doi:

    Figure Lengend Snippet: Neutralization of IL-23 and IL-1 inhibits egg-induced IL-17 production in CBA DC-T cell cocultures. A and B , DC-T cell cocultures were established in the presence or absence of eggs, anti-CD3/anti-CD28 coated beads, and the indicated blocking reagents

    Article Snippet: After 4 days, the culture supernatants were removed and assayed by ELISA for IL-17, IL-6, and IFN- γ using Ab, standards, and protocols from R & D Systems; for IL-23, using Ab, standards, and protocols from eBioscience; and for IL-5, using Ab, standards, and protocols from BD Pharmingen.

    Techniques: Neutralization, Crocin Bleaching Assay, Blocking Assay

    Exogenous IL-23 and IL-1 β enhance egg-induced IL-17 production in BL/6 DC-T cell cocultures. A–C , DC-T cell cocultures were established in the presence or absence of eggs, anti-CD3/anti-CD28 coated beads, and recombinant cytokines as described

    Journal:

    Article Title: Dendritic Cell IL-23 and IL-1 Production in Response to Schistosome Eggs Induces Th17 Cells in a Mouse Strain Prone to Severe Immunopathology 1

    doi:

    Figure Lengend Snippet: Exogenous IL-23 and IL-1 β enhance egg-induced IL-17 production in BL/6 DC-T cell cocultures. A–C , DC-T cell cocultures were established in the presence or absence of eggs, anti-CD3/anti-CD28 coated beads, and recombinant cytokines as described

    Article Snippet: After 4 days, the culture supernatants were removed and assayed by ELISA for IL-17, IL-6, and IFN- γ using Ab, standards, and protocols from R & D Systems; for IL-23, using Ab, standards, and protocols from eBioscience; and for IL-5, using Ab, standards, and protocols from BD Pharmingen.

    Techniques: Recombinant