Journal: International immunology

Article Title: IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1β by keratinocytes via the ROS-NLRP3-caspase-1 pathway.

doi: 10.1093/intimm/dxr110

Figure Lengend Snippet: Fig. 1. IL-17 and IL-22 receptors are expressed in HaCaT cells. (A) Cultured HaCaT cells were analyzed by RT–PCR to confirm the constitutive expression of IL-17RA (167 bp) and IL-22R (183 bp). (B) Surface expression of IL-17RA and IL-22R (open area) on HaCaT single-cell suspensions compared with those of the isotype controls (filled area) by flow cytometry.

Article Snippet: To block IL-22R in HaCaT cells, the cells were incubated with either rabbit anti-human IL-22R antibody (AF2770; R&D systems) or with the isotype control (AB-108-C; R&D systems) at a concentration of 50 lg ml 1 for 2 h prior to the addition of IL-22 to the cell culture. siRNA silencing of NLRP3 expression To reduce endogenous NLRP3 expression, HaCaT cells were transfected with 80 pmol of siRNA oligonucleotides (sc-45469; Santa Cruz Biotechnology) in accordance with the manufacturer’s instructions.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Expressing, Cytometry

Journal: International immunology

Article Title: IL-17 and IL-22 enhance skin inflammation by stimulating the secretion of IL-1β by keratinocytes via the ROS-NLRP3-caspase-1 pathway.

doi: 10.1093/intimm/dxr110

Figure Lengend Snippet: Fig. 5. Anti-IL-22R Ab-treated HaCaT cells down-regulate IL-1b production and result in reduced expression of the NLRP3-mediated caspase-1 activation pathway. To block IL-22R-mediated signaling, pre-treatment of HaCaT cells with 50 lg ml1 of anti-IL-22R blocking antibody for 2 h prior to IL-22 stimulation resulted in reduced NLRP3 and caspase-1 expression (A) as well as a consequent reduction in IL-1b secretion as measured by ELISA (B). The data are expressed as the means 6 SEMs (*P < 0.05). Ab, anti-IL-22R blocking antibody; IgG, isotype antibody for the negative control of IL-22R antibody.

Article Snippet: To block IL-22R in HaCaT cells, the cells were incubated with either rabbit anti-human IL-22R antibody (AF2770; R&D systems) or with the isotype control (AB-108-C; R&D systems) at a concentration of 50 lg ml 1 for 2 h prior to the addition of IL-22 to the cell culture. siRNA silencing of NLRP3 expression To reduce endogenous NLRP3 expression, HaCaT cells were transfected with 80 pmol of siRNA oligonucleotides (sc-45469; Santa Cruz Biotechnology) in accordance with the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: Transgenic mice express functional IL-22R1 on lymphocytes. (A) A schematic diagram of the IL-22R1 transgene driven by a mouse CD2 promoter. (B) Southern blot analysis of IL-22R1 transgene in founders on a C57BL/6 background. (C) Zebra plots of flow cytometric analysis for IL-22R1 expression on myeloid (CD11b+), T (CD3+), and B (CD19+) cells in peripheral blood leukocytes. (D) Western blot analysis of STAT3 phosphorylation (Tyr705) in the thymus, lymph nodes, and spleen from tg founders and nontransgenic littermates.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Transgenic Assay, Functional Assay, Southern Blot, Expressing, Western Blot

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: The IL-22R1 transgenic founders show spontaneous multiorgan inflammation. Various organs of IL-22R1 tg mice were stained (hematoxylin and eosin) and examined under the microscope. Inflammation was observed in lung (×20; A), liver (×20; B), colon (×20; C), and kidney (×20; D). Hyperplasia with granulocytic infiltrates was observed in bone marrow (×20; E) and extramedullary hematopoiesis was seen in spleen (×20; F). Arrows show leukocytic infiltrates, and insets show enlargement of the portion of the slide. (G) Total cellularity of the thymus, lymph nodes, bone marrow, spleen, and liver from IL-22R1 tg mice (n = 3) and littermate controls (n = 3). (H) Expression of proinflammatory cytokines is elevated in IL-22R1 tg mice. Cytometric bead array to assay the concentrations of an inflammatory panel of cytokines in the serum of IL-22R1 (n = 7) tg founders and nontransgenic littermates (n = 9). IL-22 production in the serum of IL-22R1 tg and wt mice measured by ELISA (n = 6). The data are represented as the mean ± SEM. *P < .05, **P < .01, ***P < .001; ns, not significant. Images were acquired with an Olympus BX40 light microscope using Image Proplus 6 software.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Transgenic Assay, Staining, Microscopy, Expressing, Enzyme-linked Immunosorbent Assay, Light Microscopy, Software

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: Transplantation of IL-22R1 tg hematopoietic cells into wt recipients results in T cell–mediated spontaneous multiorgan inflammation. (A) Schematic diagram showing serial bone marrow transplantation in tg (IL-22R1→wt) and wt (wt→wt) recipients. (B) Total cellularity of hematopoietic organs in wt (wt→wt) and tg (IL-22R1→wt) recipients. Gross morphology of the thymus (C), spleen (D), and lymph nodes (E). Images were acquired using a digital SLR camera (Sony DSLR-A330) with a Macro 2.8/50 lens. (F) Analysis of the concentrations of inflammatory cytokines (by CBA) and IL-22 (by ELISA) in the serum of IL-22R1 tg (IL-22R1→wt) and wt (wt→wt) recipients (n = 7). The data are represented as the mean ± SEM. *P < .05, **P < 0.01, ***P < .001. (G) Kaplan-Meier survival plot of wt and Foxn1nu recipients receiving bone marrow from IL-22R1 tg mice. (H) Percentage of neutrophils (NE), lymphocytes (LY), monocytes (MO), eosinophils (EO), and basophils (BA) in peripheral blood lymphocytes from athymic nude mice compared with wt controls receiving IL-22R1 tg bone marrow.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Transplantation Assay, Enzyme-linked Immunosorbent Assay

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: IL-22R1 tg mice exhibit neutrophilia. (A) Forward scatter/side scatter plot shows that the percentages of GR1+CD11b+ population was higher in IL-22R1 tg mice compared with the wt mice. The Gr1+CD11b+ population was further distinguished based on the FSC and F4/80 gates. (B) Percentage of neutrophils (FSChiF4/80lo) and monocytes (FSC loF4/80int) from polymorphonuclear leukocytes. (C-D) Percentage of cells that are Gr1+CD11b+ in bone marrow and spleen (n = 3), respectively. (E-F) Representative cytospins of 1 × 104 cells from bone marrow (×20; E) and spleen (×20; F) stained with Giemsa. Images were acquired with an Olympus BX40 light microscope using Image Proplus 6 software.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Staining, Light Microscopy, Software

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: G-CSF, IL-22, and IL-17 are elevated in IL-22R1 tg mice. (A) Zebra plot showing intracellular levels of IL-17 and IL-22 in T cells in lungs when injected with Brefeldin A for 6 hours intravenously. Graphs showing levels of IL-17 and IL-22 as percentage (B) and total cellularity (C). (D) Histogram of flow cytometric analysis of splenocytes stimulated with PMA and ionomycin for 5 hours blocked with Brefeldin A and stained with CD4 and IL-17 antibodies. (E) Cytometric bead array to assay the concentrations of G-CSF in the serum of IL-22R1 (n = 5) tg founders and nontransgenic littermates (n = 5). The data are represented as the mean ± SEM. **P < .01.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Injection, Staining

Journal: Blood

Article Title: A novel role for IL-22R1 as a driver of inflammation

doi: 10.1182/blood-2010-05-285908

Figure Lengend Snippet: ALK+ALCL cell line characterization and leukemia model amplify IL-22, which correlates with tumor burden. (A) Histogram of flow cytometric analysis of cell lines stained for IL-22R1 and pSTAT3 (Tyr705) antibodies. (B) Relative quantification of AHR, RORC, IL-22, IL-17F, and IL-26 by real-time polymerase chain reaction. (C) Zebra-plot of flow cytometry data showing intracellular levels of IL-22 with (Bref A +) or without (Bref A −) Brefeldin A, and PMA and ionomycin stimulation with Brefeldin A (P/I+ Bref A +) for 5 hours. (D) Kaplan-Meier survival plot of the Karpas299 leukemia-bearing SCID/NOD mice injected with PBS or HeFi-1. Serum levels of sIL-2Rα (E) and IL-22 (F) in Karpas299 leukemia-bearing SCID/NOD mice at day 21 after therapy.

Article Snippet: Antibodies used for lineage analysis were T cells: CD3 (17A2; eBioscience), CD4 (RM4-5; Pharmingen), CD8 (53-6.7; Biolegend), CD25 (PC61; Pharmingen), CD44 (IM7; Biolegend), CD62L (MEL-14; Biolegend); natural killer (NK) and NK-T cells: NK1.1 (PK136; Biolegend), NKp46 (29A1.4; eBioscience), CD1d tetramer (a gift from Jeff Subleski, National Cancer Institute-Frederick, NIH); myeloid population: CD11c (N418; eBioscience), Gr1 (Ly6C&G, RB6-8C5; eBioscience), CD11b (Mac1, M1/70; Pharmingen), F4/80 (BM8; eBioscience); B cells: CD19 (1D3; Pharmingen), B220 (RA3-6B2; Pharmingen), immunoglobulin M (R6-60.2; Pharmingen), immunoglobulin D (11-26; eBioscience), CD43 (1B11; Biolegend), and IL-22R1 (antibody kindly provided by Genentech USA) with their respective isotype controls.

Techniques: Staining, Real-time Polymerase Chain Reaction, Flow Cytometry, Injection