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

Article Title: IL-17C/IL-17RE Augments T Cell Function in Autoimmune Hepatitis.

doi: 10.4049/jimmunol.1600977

Figure Lengend Snippet: FIGURE 3. IL-17C expression is upregulated in autoimmune hepatitis. (A and B) WT mice were intravenously injected with 12 mg/kg Con A. (A) Relative mRNA expression of IL-17C at different time points in liver of Con A–treated WT mice, n = 5 for each group. (B and C) Representative image of liver sections from control and Con A–treated mice (B) or angioma and AIH patients (C) that were stained for IL-17C by immunohistochemistry. Scale bars, 100 mm (left and middle) and 50 mm (right), respectively. (D) Representative images of liver sections from control and Con A–treated mice (upper panel) or angioma and AIH patients (lower panel) that were stained for IL-17RE by immunohistochemistry. Scale bars, 100 mm (left and middle) and 50 mm (right), respectively. (E) IL-17C mRNA expression in different cell types isolated from liver of normal (n = 4) or Con A–treated mice (n = 6) at 8 h post injection. (F) IL-17C mRNA expression in liver of WTand Rag12/2 mice 8 h after Con A injection, n = 4–5 mice per group. (G) IL-17C mRNA expression in mouse primary hepatocytes treated for 4 h with IL-6 (20 ng/ml), IL-4 (20 ng/ml), IL-1a (10 ng/ml), IL-17A (50 ng/ml), IFN-g (20 ng/ml), IL-1b (10 ng/ml), TNF-a (10 ng/ml). Data shown are representative of two independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Immunohistochemical staining was performed using a rabbit antimouse IL-17C polyclonal Ab (Bioss) or a goat anti-human IL-17C polyclonal Ab (R&D), rabbit anti-mouse/human IL-17RE polyclonal Ab (Biorbyt), and then incubated with a HRP-labeled secondary Ab and followed by 3,39-diaminobenzidine staining.

Techniques: Expressing, Injection, Control, Staining, Immunohistochemistry, Isolation

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

Article Title: IL-17C/IL-17RE Augments T Cell Function in Autoimmune Hepatitis.

doi: 10.4049/jimmunol.1600977

Figure Lengend Snippet: FIGURE 5. IL-17C is required for T and NK cell activation in Con A–induced hepatitis. WT and IL-17C2/2 mice were injected with 12 mg/kg Con A intravenously, and liver MNCs were isolated 24 h later. (A–F) Activation of liver CD4+

Article Snippet: Immunohistochemical staining was performed using a rabbit antimouse IL-17C polyclonal Ab (Bioss) or a goat anti-human IL-17C polyclonal Ab (R&D), rabbit anti-mouse/human IL-17RE polyclonal Ab (Biorbyt), and then incubated with a HRP-labeled secondary Ab and followed by 3,39-diaminobenzidine staining.

Techniques: Activation Assay, Injection, Isolation

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

Article Title: IL-17C/IL-17RE Augments T Cell Function in Autoimmune Hepatitis.

doi: 10.4049/jimmunol.1600977

Figure Lengend Snippet: FIGURE 7. IL-17C–dependent liver damage re- quires NK cells. (A) Representative FACS of liver NK cells from WT (n = 7) and IL-17C2/2 (n = 9) mice 24 h after Con A treatment. (B) Real-time RT- PCR analysis of TRAIL mRNA level in sorted liver CD4+T, CD8+T, NK, NKT cells of WT and IL-17C2/2

Article Snippet: Immunohistochemical staining was performed using a rabbit antimouse IL-17C polyclonal Ab (Bioss) or a goat anti-human IL-17C polyclonal Ab (R&D), rabbit anti-mouse/human IL-17RE polyclonal Ab (Biorbyt), and then incubated with a HRP-labeled secondary Ab and followed by 3,39-diaminobenzidine staining.

Techniques: Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Unveiling the Ability of Witch Hazel ( Hamamelis virginiana L.) Bark Extract to Impair Keratinocyte Inflammatory Cascade Typical of Atopic Eczema

doi: 10.3390/ijms23169279

Figure Lengend Snippet: The effect of HVE and HT treatment (24 h) on the release of IL-17C ( a , b ) and MMP-9 ( c , d ) by HaCaT cells challenged by TNF-α (10 ng/mL), measured by the ELISA assay. HT was not effective until the highest concentration tested (10 μM corresponding to 4.93 μg/mL). The data are expressed in percentages, relative to the stimulated control, which is arbitrarily assigned a value of 100%. ** p < 0.01, *** p < 0.001 versus stimulus. HVE, Hamamelis virginiana bark extract; reference inhibitor: E, epigallocatechin gallate (20 μM).

Article Snippet: Coating anti-human IL-17C antibody and biotinylated anti-IL-17C were purchased from Novus (Novus biologicals, Bio-Techne s.r.l., Milan, Italy).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: Recurrent HSV-2 infection induces IL-17c expression in keratinocytes. (A) Isolation of keratinocytes above basement membrane by LCM. (B) Expression of keratin 5 (KRT5) and 14 (KRT14), CD1a and CD8a in laser-captured keratinocytes (kera), CD1a + Langerhans cells (LC), and CD8a + CD8 T cells (CD8) from control (ctrl) and posthealed (PH) genital skin biopsies during recurrent HSV-2 infection. Y-axis, intensity values from normalized Illumina bead array data. The displayed values are the means for keratinocytes ( n = 4), CD1a + Langerhans cells ( n = 8), and CD8a + CD8 T cells ( n = 8). 1, kera_ctrl; 2, kera_PH; 3, CD8_ctrl; 4, CD8_PH; 5, LC_ctrl; and 6, LC_PH. (C) HSV infection in keratinocytes induced IL-17c expression in vivo. Comparison of expression of cytokines/chemokines (top) and six different members of IL-17 (bottom) in keratinocytes isolated from lesion and posthealed skin (asymptomatic shedding) biopsies to those from contralateral control biopsies. (D) IL-17c protein expression in epidermal keratinocytes in skin biopsies during lesion and shedding (clinical quiescence 8-wk shedding). IL-17c expression was detected by immunofluorescent staining with an anti-IL-17c antibody (green) and nuclei stained with DAPI (blue). Bar, 50 µm.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Infection, Expressing, Isolation, Membrane, Control, In Vivo, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: HSV-2 infection in human primary keratinocytes induces IL-17c, which does not appear to have significant antiviral effect. (A) IL-17c expression in HSV infected human primary keratinocytes. IL-17c RNA expression in a time course of HSV-2-infected keratinocytes (left). Cells were mock infected (aqua) or infected with HSV-2 (HG52; MOI = 1) in the absence (blue) or presence (green) of acyclovir (30 µM) or with UV-inactivated viruses (red). Purple is acyclovir without virus. Y-axis is fold change of RNA levels above mock infected cells; x-axis is time after infection in hours. HSV-2 virus growth curve in primary keratinocytes was determined by plaque assay (middle). Y-axis is PFU (plaque forming units) per million cells; x-axis is time post-infection (hours) of HSV-2 in the absence (blue) or presence (green) of acyclovir or UV-inactivated viruses (red). IL-17c expression in a time course of HSV-1-infected human primary keratinocytes (right). Cells were mock infected or infected with HSV-1 (KOS; MOI of 1). (B) IL-17c protein expression in HSV-2-infected keratinocytes. Cells mock (bottom) or HSV-2 infected (top) for 7 h at MOIs of 1 and 10 were analyzed for IL-17c expression by immunofluorescent staining with an anti–IL-17c antibody (red). Nuclei stained with DAPI (blue). Graph is quantification of staining as the percentage of IL-17c-expressing cells. M, mock; 1, MOI of 1; 10, MOI of 10. Bar, 50 µm. (C and D) HSV-2 and TLR ligands additively induced IL-17c expression in human primary keratinocytes. Cells were treated with TLR ligands (PGN, poly[I:C]/LyoVec, poly[I:C], LPS, flagellin, and ODN) for 2, 6, and 24 h (C) or infected with HSV-2 for 3 h and then treated with poly[I:C]/LyoVec, poly[I:C], or flagellin for another 4 h (D). Y-axis, IL-17c RNA expression fold changes relative to untreated and mock infected controls. (E) Blocking IL-17c signaling does not have significant effect on HSV-2 gene expression or viral titers in infected human primary keratinocytes. Cells were pretreated with an IL-17RA neutralizing antibody (red) or matching control IgG (blue) for 1 h before HSV-2 infection. Gene expression ( ICP27 ) was assayed by quantitative PCR (left), and viral titers were determined by plaque assay in Vero cells (right). Error bars represent 1 standard deviation from the mean of three replicates. All the experiments were repeated three times.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Infection, Expressing, RNA Expression, Virus, Plaque Assay, Staining, Blocking Assay, Gene Expression, Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: Peripheral nervous systems express IL-17RE, a receptor subunit specific for IL-17c. (A) Nerve endings in lesional genital skin expressed IL-17RE. IL-17RE + cells exhibited elongated fiber-like shapes and were distinct from CD15 + , CD8a + , and CD4 + cells. Double immunofluorescent staining with anti-IL-17RE (red) and anti-CD15, CD8a or CD4 (green) antibodies revealed no costaining (left). Double immunofluorescent staining with anti-NCAM (green) and anti-IL-17RE (red) antibodies showed significant double staining of nerve endings in lesional biopsies (right). Nuclei stained with DAPI (blue). Bar, 50 µm. (B) Nerve endings in control skin biopsies express IL-17RE and IL-17RA. Double staining with anti-NCAM and anti-IL17RE or IL-17RA antibody showed double-positive nerve endings in control skin biopsies. Bar, 50 µm. (C) Nerve endings in posthealed skin biopsies express IL-17RE and IL-17RA. Double immunofluorescent staining with anti-peripherin (green) and anti-IL-17RE (red) antibodies (left) or anti-NCAM (green) and anti-IL17RA (red) antibodies (right) revealed expression of IL-17RE on peripherin + nerve endings and expression of IL-17RA on NCAM + nerve endings in genital 4-wk posthealed skin biopsies. Nuclei stained with DAPI (blue). Bar, 50 µm. (D) Single immunofluorescent staining with anti-IL-17RE (red) in sensory neurons from human fetal DRG showed staining in both neuronal cell bodies and nerve fibers. Insets show enlarged pictures of IL-17RE expression in cell bodies (top) and axons (bottom). Bar, 500 µm. (E) Detection of IL-17RE RNA expression in sensory neurons in human fetal DRG using FISH. TUBB3, tubulin β 3 class III. Three representative images are displayed. Bars, 50 µm. (F) IL-17RE (red) expression in a subset of NF200 + (left; green) or peripherin + neurons (right; green) and axons in human fetal DRG. Bar, 50 µm. All the experiments were repeated three times.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Staining, Double Staining, Control, Expressing, RNA Expression

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: IL-17c stimulated neurite growth of differentiated SY5Y neurons. (A) IL-17c induced directional neurite growth of differentiated SY5Y cells in a microfluidic device. SY5Y cells were differentiated with all-trans retinoid acids (ATRA) at 20 µg/ml for 4 d and then placed in the wells on the left side of a microfluidic device with culture medium alone (M) or medium plus IL-17c, NGF, or BDNF with or without neutralizing antibodies for IL-17RA (α-IL17RA), NGF (α-NGF), or BDNF (α-BDNF), respectively, on the other side. After 10 d of culture, cells were fixed and stained with a PGP9.5 antibody. Bar, 200 μm. (B) Comparison of total length of neurites and numbers of neurites from all the conditions shown in A. P-value is derived from two-sample t test with unequal variance ( n = 3). *, P = 0.047; **, P = 0.036. Error bars represent 1 standard deviation from the mean of three replicates. (C) Size-exclusion chromatogram (SEC) showing an overlay of the soluble forms of human IL-17c (residues 19–197), human IL-17RE (residues 155–451), and human IL-17RA (residues 33–317). Each construct runs as a well-behaved, monodispersed protein of appropriate molecular weight. Gel inset shows a fraction corresponding to each peak analyzed under nonreducing, SDS-PAGE conditions (top). SEC supershift assay showing that recombinant human IL-17c interacts both as ternary complex with IL-17RA and IL-17RE and as a binary complex with IL-17RE alone. Gel inset shows a fraction corresponding to each peak analyzed under nonreducing, SDS-PAGE conditions (bottom). All the experiments were repeated three times.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Staining, Comparison, Derivative Assay, Standard Deviation, Construct, Molecular Weight, SDS Page, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: IL-17c induced neurite growth and branch points in HSNs. HSNs were isolated from fetal spinal tissue and cultured in full neural medium only or medium plus IL-17c or NGF. (A) Images of cultured HSNs in the presence of IL-17c at 75 h (top) and 90 h (bottom) after plating. Bar, 300 μm. (B) Live imaging of HSNs to measure neurite length (left graph), neurite branch points (middle graph), and cell body area (right graph) every hour for 16 h from hours 75 to 90 after HSNs were plated in culture medium or medium plus IL-17c or NGF. (C) Growth rates of neurite length (left graph), neurite branch points (middle graph), and cell body area (right graph) of cultured HSNs from hours 75 to 90 after HSNs were plated. (D) A microfluidic device with three channels. HSNs were placed in the middle channel and medium only (M) and medium plus IL-17c was placed on the left and right channels, respectively. DRG, dorsal root ganglia. (E and F) HSNs extended significantly longer neurites with more branch points into the channel with IL-17c containing medium. HSNs were fixed and stained with PGP9.5 after 16 d of culture (E) and number of neurites, total length and branch points were counted (F). Bar, 500 µm. (G and H) The HSN neurites expressed IL-17RE. HSNs in the three-channel device were double-stained with PGP9.5 and IL-17RE antibodies (G). Comparison of growth cones of neurites from medium only and IL-17c-containing channels (H). Bars, 50 μm (H), 10 μm (G). HSN experiments were repeated three times using different fetal DRG.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Isolation, Cell Culture, Imaging, Staining, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Keratinocytes produce IL-17c to protect peripheral nervous systems during human HSV-2 reactivation

doi: 10.1084/jem.20160581

Figure Lengend Snippet: IL-17c pretreatment reduces apoptosis during HSV-2 infection of MCNs. (A) HSV-2 infection induces expression of IL-17c in MCNs. Cells were infected with HSV-2 (186) at MOI of 5 for 1, 3, 6, 12, 24, and 48 h. Y-axis is fold change over mock infected MCNs. Gene expression (IL-17c and β-actin [ACTB]) was determined by quantitative PCR (top), and virus titers were determined by plaque assays (bottom). (B) Detection by immunofluorescence of cleaved caspase 3 levels in HSV-2 (186) infected MCNs. MCNs were untreated or pretreated with mIL-17c for 24 h in the presence of a murine IL-17RA–neutralizing antibody (anti-mIL17RA) or matching control rat IgG before HSV-2 infection at MOI of 5 for 8 h. Cells were stained with DAPI for cell nucleus, an antibody for β-tubulin III to visualize neurons (green) and an antibody for cleaved caspase 3 (red). Bar, 100 μm. (C) Percentages of cleaved caspase 3 + neurons in HSV-2 (186) infected neurons pretreated with mIL-17c (24 h) in the presence of anti-mIL17RA or control IgG (left) or pretreated with anti-IL-m17RA or control IgG (1 h; right). *, P = 0.01. (D) Detection of caspase 3/7 enzymatic activities in HSV-2 (186) infected MCNs. MCNs were treated and infected similarly as described in B and caspase 3/7 enzymatic activities were assayed by luminescence (relative light unit [RLU]). Error bars represent 1 standard deviation from the mean of three replicates. P-value is derived from two-sample t test with unequal variance. **, P = 0.05. The experiments were repeated three times.

Article Snippet: The antibodies for staining were purchased from the following sources: IL-17c antibody (mouse monoclonal, R&D Systems); IL-17RE antibody (rabbit polyclonal, Sigma-Aldrich); IL-17RA antibody (rabbit monoclonal, LifeSpanBioSciences); NCAM antibody (mouse monoclonal, BD); PGP9.5 (Abcam); NF200 antibody (rabbit polyclonal, Sigma-Aldrich); Peripherin antibody (rabbit and mouse, Sigma-Aldrich); cleaved caspase 3 antibody (rabbit polyclonal, Cell Signaling Technology).

Techniques: Infection, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Virus, Immunofluorescence, Control, Staining, Standard Deviation, Derivative Assay