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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, IL-17RA and IL-17RC mRNA expression was determined by RT-PCR in RAW264.7 cells (positive control), and primary astrocytes, in duplicate. B, Primary astrocytes were treated with medium (UN), IL-6/R (IL-6, 10 ng/ml and sIL-6R, 25 ng/ml), IL-17 (25 ng/ml) or IL-6/R plus IL-17 for up to 24 h, and levels of IL-6 and GAPDH mRNA expression were determined by RT-PCR and QRT-PCR. C, Primary astrocytes were treated with medium (UN), IL-6/R, different concentrations of IL-17 (1-50 ng/ml) or IL-6/R + IL-17 (1-50 ng/ml) for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. D, Primary astrocytes were treated with medium (UN), IL-6, sIL-6R or IL-17 in various combinations for 4 h, and levels of IL-6 mRNA expression were determined by QRT-PCR. E, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 48 h, and supernatants were analyzed for IL-6 protein using ELISA. Experiments shown are representative of at least three experiments. All data are the mean ± SD of three experiments. *, p ≤0.05; **, p ≤0.01.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against IκBα and GAPDH. The basal level of the untreated sample was set at 100, and the percentage change of IκBα upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment compared with the basal value. B, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65 and GAPDH. C, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 15 and 30 min, and then cell lysates were immunoblotted with antibodies against phospho-ERK1/2, ERK1/2 and GAPDH. D, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R and IL-17 for 30 min and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-p38, p38 and GAPDH. E, Astrocytes were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 30 and 60 min, and then cell lysates were immunoblotted with antibodies against phospho-JNK, JNK and GAPDH. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R + IL-17 treatment was compared with that value (B, C, D and E). Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Activation Assay

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were cultured in absence or presence of IL-17 followed by IL-6/R treatment for 4 h. Actinomycin D (5 ng/ml) was then added, and cells harvested at 0, 30, 60, 120, and 240 min after addition. The abundance of IL-6 mRNA was determined by QRT-PCR. B, C, DMSO vehicle, BAY 11 (5 μM), U0126 (10 μM), SB203580 (10 μM) or JNKi II (10 μM) were added to cultures 1 h before cytokine addition, and then astrocytes were incubated with medium, IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h. Levels of IL-6 mRNA expression were determined by QRT-PCR. All data are the mean ± SD of three experiments. **, p ≤ 0.01; NS = not significant.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Quantitative RT-PCR, Incubation, Expressing

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Primary astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 90 min, and then cells were cross-linked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with Abs against p65, P-p65, c-Fos, c-Jun, or normal rabbit IgG. PCR analysis of the positive control (input) indicates that soluble chromatin samples obtained from each time point had equal amounts of chromatin fragments containing the IL-6 promoter. B, Primary astrocytes were treated as above. Soluble chromatin was subjected to immunoprecipitation with Abs against histone acetylation (Ac-H3 and Ac-H4), p300, CBP, RNA Pol II or normal rabbit IgG. The basal level of the untreated sample was set at 1.0 and fold activation upon IL-6/R, IL-17 or IL-6/R plus IL-17 treatment was compared with that value. Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Immunoprecipitation, Positive Control, Activation Assay

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, Astrocytes were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 2 - 8 h, and levels of SOCS3 mRNA expression were determined by QRT-PCR. B and C, Astrocytes were transfected with SOCS3 siRNA (100 nM) or siRNA control (100 nM) for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 4 h. Levels of SOCS3 (B) and IL-6 (C) mRNA expression was determined by QRT-PCR. D, Astrocytes were transfected with SOCS3 siRNA or siRNA control for 48 h. Transfected cells were then treated with medium (UN) or IL-6/R plus IL-17 for 24 h, and supernatants analyzed for IL-6 protein by ELISA. E, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were harvested. Digestion of genomic DNA distinguishes the full-length (fl) and excised alleles (Δ). F, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, the cells were treated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 4 h, and levels of IL-6 mRNA expression determined by QRT-PCR. G, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were incubated with medium (UN), IL-6/R, IL-17 or IL-6/R plus IL-17 for 16 or 24 h, and supernatants analyzed for IL-6 protein by ELISA. All data are the mean ± SD of three experiments. *, p ≤ 0.05;**, p ≤ 0.01.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Infection, Incubation

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: To evaluate NF-κB and MAPK activation in the absence or presence of SOCS3, SOCS3 floxed astrocytes were infected with GFP as control or GFP-Cre for deletion. After 48 h in culture, cells were treated with medium (UN) or IL-6/R plus IL-17 for 15, 30, 60 or 120 min, and then cell lysates were immunoblotted with antibodies against phospho-p65 Ser 536, p65, phospho-p38, p38, phospho-JNK, JNK or GAPDH. The basal level of the untreated GFP infected sample was set at 1.0 and fold activation upon IL-6/R plus IL-17 treatment compared with that value. Representative of at least three experiments.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Infection

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

Article Title: Interleukin-17 Enhancement of the Interleukin-6 Signaling Cascade in Astrocytes

doi: 10.4049/jimmunol.1000142

Figure Lengend Snippet: A, IL-6/R and IL-17 activate the NF-κB and MAPK pathway, which then induce IL-6 gene expression. The synergistic effect of these two mediators depends on NF-κB, p38 and JNK MAPK activity. Activated NF-κB p65, c-Fos and c-Jun bind to the IL-6 promoter. Concurrent with NF-κB and MAPK recruitment, IL-6/R and IL-17 leads to the recruitment of coactivators CBP and p300, modifications in AcH3 and AcH4, and recruitment of RNA Pol II to the IL-6 promoter, which results in transcriptional activation of the IL-6 gene. IL-17 can also enhance IL-6/R induced SOCS3 expression, and SOCS3 inhibits IL-6/R plus IL-17-induced NF-κB and MAPK activation, which results in a reduction of IL-6 gene expression in astrocytes. B, Naive CD4+ T cells, after activation by signaling through the T cell receptor and co-stimulatory molecules, can differentiate into Th17 cells in the presence of IL-6, TGF-β, IL-1 and IL-23. IL-17 together with IL-6/R triggers a positive-feedforward loop of IL-6 expression in astrocytes, which may also influence Th17 cell differentiation. SOCS3 participates in these processes as a negative feedback regulator. See text for details.

Article Snippet: Recombinant human IL-6, recombinant human soluble IL-6 Receptor (sIL-6R) and recombinant mouse IL-17 were from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Activity Assay, Activation Assay, Cell Differentiation

Journal: Science Advances

Article Title: Pregnancy enhances antiviral immunity independent of type I IFN but dependent on IL-17–producing γδ + T cells in the nasal mucosa

doi: 10.1126/sciadv.ado7087

Figure Lengend Snippet: ( A ) Model of murine intratracheal IAV infection (50 PFU in 50 μl of PBS). Created with BioRender.com . ( B ) Kinetics of pulmonary viral titers by MDCK plaque assay following intratracheal IAV infection ( n = 7 to 9). ( C ) Graphical representation of upper airway infection (50 PFU IAV in 10 μl of PBS). Created with BioRender.com . ( D ) Kinetics of the viral load measured by MDCK plaque assay on nasal tissues from WT nonpregnant and pregnant mice ( n = 7 to 10). ( E ) Graphical representation of nasal tissue regions and fluorescence microscopy of infected olfactory epithelial cells (zone III) with quantification 3 days postinfection with Ruby-NS1 PR8 (500 PFU in 10 μl of PBS) ( n = 3 to 4). Scale bars, 200 μm. ( F ) Kinetics of viral load measured by MDCK plaque assay on nasal tissues of nonpregnant and pregnant Ifnar1 −/− mice (50 PFU; n = 4 to 7). ( G ) Kinetics of nasal il-17A expression by RT-qPCR (50 PFU; n = 4 to 5). ( H ) Representative FACS plots and frequency of γδ + T cells, ( I ) absolute number of γδ + T cells, ( J ) proportion of IL-17A–producing cells and ( K ) total number of IL-17A + γδ + T cells in nasal tissues of nonpregnant and pregnant mice at 0 and 1 day post-IAV infection following 4 hours of stimulation with rIL-23 (10 ng/ml) and rIL-1β (10 ng/ml) (50 PFU; n = 7 to 10). ( L ) Ns1 and ( M ) M1 expression in nasal tissues of WT and TCR δ −/− pregnant mice by RT-qPCR 3 days post-IAV infection (50 PFU; n = 5). ( N ) Ns1 and ( O ) M1 expression in nasal cavities of TCR δ −/− nonpregnant and pregnant mice 3 days post-IAV infection ( n = 5 to 8). Data produced from . Data are presented as mean ± SEM. with * P < 0.05, ** P < 0.01, and *** P < 0.001. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons [(B), (D), and (F) to (K)], unpaired Student’s t test [(E), (L), (N), and (O)], or Mann-Whitney U test (M).

Article Snippet: Nonpregnant mice were administered rIL-17A (1 μg per mouse; R&D Systems, #7956-ML-025/CF) intranasally in 10 μl of PBS (5 μl per naris) 2 hours before IAV infection (50 PFU intranasally).

Techniques: Infection, Plaque Assay, Fluorescence, Microscopy, Expressing, Quantitative RT-PCR, Produced, MANN-WHITNEY

Journal: Science Advances

Article Title: Pregnancy enhances antiviral immunity independent of type I IFN but dependent on IL-17–producing γδ + T cells in the nasal mucosa

doi: 10.1126/sciadv.ado7087

Figure Lengend Snippet: ( A to E ) Muc5ac , Cramp , Mbd-3 , Mbd-4 , and Mbd-14 , expression in the nasal cavities of nonpregnant and pregnant IAV-infected mice measured by RT-qPCR at various time points postinfection (50 PFU; n = 4 to 5). ( F ) Loading plot and correlation coefficients of Ns1 and AMP expression by RT-qPCR 3 days post-IAV infection ( n = 4 to 5). ( G ) Intraperitoneal (i.p.) administration of anti-IgG1 (αIgG1) or anti–IL-17A (αIL-17A) (200 μg per mouse in 200 μl of PBS) to nonpregnant and pregnant mice 2 days before and the day of IAV infection (50 PFU in 10 μl). Created with BioRender.com . ( H to K ) Quantification of nasal Muc5ac , Cramp , Mbd-3 , and Mbd-4 expression by RT-qPCR ( n = 10 to 11) and ( L ) nasal viral titers quantified by MDCK plaque assay 1 day post-IAV infection following intraperitoneal administration of αIL-17A ( n = 7 to 8). ( M to O ) Neutrophil, monocyte, and macrophage absolute numbers in the nasal cavities of nonpregnant and pregnant mice at days 0 and 1 post-IAV infection measured by flow cytometry (50 PFU; n = 7 to 11). Viral ( P ) Ns1 and ( Q ) M1 gene expression measured by RT-qPCR in murine epithelial cells 24 hours post-IAV infection (MOI of 1) with/without recombinant mBD-3 (rmBD-3; 100 μg/ml) ( n = 3). ( R ) Intranasal administration of rmBD-3 (1 μg per mouse; in 10 μl PBS) 1 hour post-IAV infection (50 PFU in 10 μl of PBS) to nonpregnant mice. Created with BioRender.com . Viral ( S ) Ns1 and ( T ) M1 gene expression measured by RT-qPCR in nasal tissues of nonpregnant mice 1 day post-IAV infection with/without rmBD-3 ( n = 7 to 8). Data are presented as mean ± SEM. with * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Data were analyzed using two-way ANOVA followed by Sidak’s multiple comparisons [(A) to (E) and (M) to (O)], Pearson’s correlation (F), unpaired Student’s t test [(H), (K), (L), (P), and (Q)], or Mann-Whitney U test [(I), (J), (S), and (T)].

Article Snippet: Nonpregnant mice were administered rIL-17A (1 μg per mouse; R&D Systems, #7956-ML-025/CF) intranasally in 10 μl of PBS (5 μl per naris) 2 hours before IAV infection (50 PFU intranasally).

Techniques: Expressing, Infection, Quantitative RT-PCR, Plaque Assay, Flow Cytometry, Recombinant, MANN-WHITNEY