Journal: Immunity

Article Title: Interleukin-17B antagonizes interleukin-25-mediated mucosal inflammation

doi: 10.1016/j.immuni.2015.03.008

Figure Lengend Snippet: (A) Weight loss (% of starting weight) was assessed daily in WT and IL-17B-deficient mice following DSS administration. n = 5-6 mice per group. Colons were isolated from individual mice following sacrifice at d 8 and then measured and combined for each group (B). (C + D) Colon tissue derived from the mice in (A) were sectioned and then stained with H&E to measure infiltration and inflammation in individual mice following DSS onset at d 8. (E) Total colon tissue was divided into similar sections and then combined for each mouse. mRNA was then isolated and gene values were measured by qPCR using the expression of Actb as a reference. Supernatants from the colon were also assayed for cytokine expression by ELISA. Data are presented as mean + SD and are representative of at least 3 independent experiments. *Student’s t test; p < 0.05 in comparisons of IL-17B-deficient to WT controls. See also Figure S2.

Article Snippet: Primary CEC were plated and rested in 96-well dishes prior to stimulation with LPS (Sigma-Aldrich), Pam3CSK4 (Invivogen), MDP (Invivogen), or the indicated concentration of mouse IL-17B and/or IL-25 (R & D Systems) for 6h (mRNA) or 24h (ELISA).

Techniques: Isolation, Derivative Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Immunity

Article Title: Interleukin-17B antagonizes interleukin-25-mediated mucosal inflammation

doi: 10.1016/j.immuni.2015.03.008

Figure Lengend Snippet: (A) Bone marrow chimeric mice were generated using WT and Il17rb−/ animals as donors and recipients. The first portion of the legend represents hematopoietic cell origin (WT or Il17rb−/−) while the second represents non-hematopoietic cell origin. (B) IL-6 expression was measured by qPCR following 6 h stimulation with recombinant IL-25 in a colon epithelial cell line (YAMC) and primary colon epithelial cells (CEC). (C+D) IL-6 mRNA expression was similarly measured from CEC derived from IL-17RB- (C) and Act1 (Traf3ip2−/−) - (D) deficient mice. (E) IL-6 was examined in CEC following fixed IL-25 stimulation (200ng/ml) followed by simultaneous treatment of increasing IL-17B concentrations. IL-6 was measured by qPCR (6 h stimulation, top) and ELISA (24 h stimulation, bottom). (F) Summary of competition experiments measuring the ability of increasing concentrations of IL-17B or IL-17A to displace IL-25-hIg bound to murine IL-17RB (RB) or IL-17RB and IL-17RA (RB+RA). Competition (%) is the percentage of IL-25 displacement at each IL-17B or IL-17 concentration compared those treated with only IL-25. (G) Combined weight loss data from WT, IL-17B-deficient, and IL-17B and IL-17E double deficient (IL-17B-deficient,Il25−/−) mice following DSS administration. n = 7-8 mice per group. (H) Representative H & E histology and combined histological scores of all colon sections from the mice shown in (G). (I) Combined IL-6 mRNA expression from total colon samples derived from the mice in (G). Data are presented as mean + SD and are representative of 2 independent experiments (A) and at least 3 independent experiments (B-I). mRNA data was obtained by examining relative gene expression to Actb. *Student’s t test; *p < 0.05 for daily comparisons of WT:WT mice to Il17rb−/−: Il17rb−/− mice and **p < 0.05 for daily comparisons of WT: Il17rb−/− mice to Il17rb−/−: Il17rb−/− mice (A); *p < 0.05 for daily comparisons of IL-17B-deficient,Il25−/− mice to WT (G); **p < 0.05 for comparison of IL-17B-deficient,Il25−/− mice to IL-17B-deficient and to WT (G-I). See also Figure S3.

Article Snippet: Primary CEC were plated and rested in 96-well dishes prior to stimulation with LPS (Sigma-Aldrich), Pam3CSK4 (Invivogen), MDP (Invivogen), or the indicated concentration of mouse IL-17B and/or IL-25 (R & D Systems) for 6h (mRNA) or 24h (ELISA).

Techniques: Generated, Expressing, Recombinant, Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

Journal: Immunity

Article Title: Interleukin-17B antagonizes interleukin-25-mediated mucosal inflammation

doi: 10.1016/j.immuni.2015.03.008

Figure Lengend Snippet: (A) WT and IL-17B-deficient, WT and IL-25KO (C), or WT and IL-17B-deficient,Il25−/− mice (E) mice were orally infected with 2 × 109 CFU of Citrobacter rodentium and weighed daily for 12-13 d. n = 7-18 mice per group. (B-F) After harvest, feces were collected, homogenized, and then examined for Citrobacter rodentium growth on MacConkey agar after overnight incubation. Data are presented as mean + SD and are representative of at least 3 independent experiments. *Student’s t test; p< 0.05. See also Figure S4.

Article Snippet: Primary CEC were plated and rested in 96-well dishes prior to stimulation with LPS (Sigma-Aldrich), Pam3CSK4 (Invivogen), MDP (Invivogen), or the indicated concentration of mouse IL-17B and/or IL-25 (R & D Systems) for 6h (mRNA) or 24h (ELISA).

Techniques: Infection, Incubation

Journal: Immunity

Article Title: Interleukin-17B antagonizes interleukin-25-mediated mucosal inflammation

doi: 10.1016/j.immuni.2015.03.008

Figure Lengend Snippet: (A) Total cell counts in the BAL fluid of WT, IL-17B- and IL-25-deficient animals sensitized with OVA in alum at day 0 and 14, followed by intranasal challenge with OVA at day 14, 25, and 26. (B) BALF derived from the animals in (A) was assayed for the presence of IL-4, IL-5, and IL-13 by ELISA. (C) Combined mRNA analysis of Th2 and Th9 cell-associated genes from total lung samples derived from the mice in (A). (D) Representative (left) and combined analysis (right) of Th2 cell frequencies in the BALF of OVA and alum sensitized animals. T cells isolated from the BALF were restimulated with 50μg/ml OVA prior to intracellular cytokine staining for IL-4 and IL-5. (E) Analysis of splenic and mediastinal lymph node Th2 compartments following 3 d restimulation with increasing doses of OVA. IL-4, IL-5, and IL-13 protein was measured from the supernatants by ELISA. Data are presented as mean of data obtained from individual mice + SD and are representative of at least 3 independent experiments. *Student’s t test; *p < 0.05 for comparisons of WT to IL-17B-deficient and **p < 0.05 for comparisons of WT to Il25−/−. See also Figure S5.

Article Snippet: Primary CEC were plated and rested in 96-well dishes prior to stimulation with LPS (Sigma-Aldrich), Pam3CSK4 (Invivogen), MDP (Invivogen), or the indicated concentration of mouse IL-17B and/or IL-25 (R & D Systems) for 6h (mRNA) or 24h (ELISA).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Isolation, Staining