Fig 1.
IL-23p19 and EBI3 proteins interact and form a stable, secreted heterodimeric complex.
(A) Co-expression of p19 and EBI3 proteins at the intracellular level. Intracellular FACS staining of HEK293FT cells that were transiently transfected with p19 or with His-tagged EBI3 plasmids alone or co-transfected with p19 and His-tagged EBI3 plasmids. Cells were fixed and permeabilized, followed by intracellular staining using p19 and EBI3-specific antibodies. (B and C) Co-immunoprecipitation studies using pre-cleared supernatants originating from tagged or untagged p19/EBI3 double transfected HEK293FT cells. Samples were subjected to anti-His Ab (B) or anti-p19 antibody (C) immunoprecipitation, followed by anti-His, p19 and EBI3 Western blotting. In addition, supernatants from transfected HEK293 FT cells were monitored for the presence of the IL-23 cytokine using a sensitive IL-23 ELISA. Representative results from at least three independent experiments are shown. (D and E) Detection of p19/EBI3-His heterodimeric complex by ELISA. Microtiter plates were coated with an anti-p19 Ab, followed by blocking. The wells were either incubated with p19/EBI3-His containing supernatants (D) or with human IL-39 IgG1-Fc fusion or IgG1-Fc control protein (E). After washing, the wells were incubated with biotinylated EBI3 Ab, followed by washing and by incubation with HRP-conjugated Avidin. (F) Specificity of IL-39 ELISA against the related IL-23 or IL-27 cytokines. Microtiter plates were coated with an anti-p19 Ab, blocked and incubated with recombinant human IL-23 or IL-27 cytokines or with the IL-39 IgG1-Fc fusion protein. After washing, the wells were incubated with biotinylated EBI3 Ab, followed by washing and by incubation with HRP-conjugated Avidin. Representative results from at least three independent experiments are shown.
Fig 2.
mRNA expression, but no protein expression of the IL-39 subunits from human immune cells and keratinocytes.
(A) Purified human B cells were incubated with medium only or stimulated with LPS (1 μg/ml) or with a combination of LPS (1 μg/ml) and BAFF 50 ng/ml for 48 hrs. (B) HaCaT cells were incubated with medium or were stimulated with the TLR3 agonist Poly IC (30 μg/ml) for 48 hrs. (C) Polarized M0, M1 or M2 macrophages were subjected to quantitative RT-PCR analysis. (A-C) mRNA was extracted and transcript levels were measured by quantitative RT-PCR. Gene expression was normalized to β-glucoronidase levels and expressed as arbitrary units. (D) Intracellular FACS staining of B cells from (A). (E and F) Supernatants originating from cultures (A-C) or an IL-39 IgG1-Fc fusion protein were used in ELISAs where anti-p19 Ab was used for capturing of p19 and anti-EBI3 served as the secondary Ab. Graphs are representative of two independent experiments.
Fig 3.
Human IL-39 does not activate immune cells and does not activate STAT3.
(A) Human neutrophils were incubated with IL-39 containing concentrated supernatants or with recombinant human IL-39 fusion or IgG1-Fc control protein (both at 1 μg/ml). After 24 hrs, the reported IL-39-controlled target BAFF and other neutrophil mediator genes were quantified by quantitative RT-PCR. Gene expression was normalized to β-glucoronidase levels and expressed as arbitrary units. Graphs are representative from two experiments containing three technical replicates each. Error bars represent the SD. (B and C) PBMCs were stimulated with CD3+CD28 Abs in the presence of IL-39 IgG1-Fc fusion or IgG1-Fc control protein (3 μg/ml). After 72 hrs of incubation, IFNγ and IL-17A concentrations present in the supernatants were determined by ELISAs. Data represent triplicate measurements of three independent experiments.
Fig 4.
Human IL-39 does not activate STAT3 and does not inhibit IL-23-induced signaling.
(A) HEK-BlueTM STAT3 IL-6 cytokine reporter cells were transiently transfected with a plasmid encoding for the human IL-23 receptor and after 48 hrs, expression of the IL-39 receptor genes IL23r and Gp130 were quantified by quantitative RT-PCR. The Ct values for IL23r and Gp130 were 22 and 26, respectively. (B) HEK-BlueTM STAT3 transfected cells from (A) were incubated with recombinant, human IL-6 (1 ng/ml), with IL-39 IgG1-Fc fusion or IgG1-Fc control protein (10 μg/ml) or with concentrated IL-39 containing supernatants originating from HEK293FT cells transfected with either p19, EBI3-His or with a combination of p19/EBI3-His. After 20 hrs, secretion of the STAT3-induced SEAP reporter was measured by QUANTI-BlueTM. (C and D) IL-39 does not antagonize IL-23-mediated signal transduction. HEK-BlueTM IL-23 cells stably expressing the human IL-23 receptor and a STAT3-inducible SEAP reporter were stimulated for 20 hrs with various concentrations of recombinant human IL-23 to induce the STAT3-mediated SEAP reporter. Alternatively, the cells were incubated with a cocktail containing IL-23 plus different concentrations of IL-39 IgG1-Fc fusion or IL-23 plus IgG1-Fc control protein. Secretion of the STAT3-induced SEAP reporter was measured by QUANTI-BlueTM.