The extracellular domain of IL-10R2 is not sufficient to maintain IL-10 activity.

<p>CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2^Δ230–330 (extracellular domain), IL-10R2^Δ1–190 (intracellular domain) or an empty vector (mcs) and cultured for 24 hours. Surface expression of IL-10R1 or IL-10R2 was analysed by flow cytometry and mean fluorescent intensity is plotted (<i>n</i> = 6, error bars represent standard error) (A). Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (100 ng/ml) in CHO-K1 cells upon transient transfection with IL-10 receptors was analysed by western blot (B).</p

The intracellular domain of IL-10R2 mediates conformational changes in IL-10R1.

<p>CHO-K1 cells were co-transfected with combinations of the expression vectors for IL-10R1, IL-10R2, IL-10R2^Δ230–330, IL-10R2^Δ1–190 or an empty vector (mcs) and cultured for 24 hours. Cells were surface stained with the 1B1.3a anti-mouse IL-10R1 monoclonal antibody and analysed by flow cytometry (A). Mean fluorescent intensity for IL-10R1 binding is plotted in a dose-dependent manner (<i>n</i> = 6, error bars represent standard error). IL-10 was cross-linked to the surface of transfected cells and surface-bound IL-10 was detected by flow cytometry (B). Mean fluorescent intensity for IL-10 binding is plotted (<i>n</i> = 3, error bars represent standard error). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

Tyk2 affects early responses to IL-10.

<p>Bone marrow-derived macrophages and dendritic cells from Tyk2^-/- mice were investigated in more detail for their signaling. Phosphorylation of tyrosine 705 (Y705) of STAT3 by IL-10 (0, 1, 10 and 100 ng/ml) was analysed in wild-type and Tyk2^-/- macrophages and dendritic cells by western blot (A and B respectively). Relative up-regulation of SOCS3 mRNA expression by IL-10 was analysed by quantitative PCR in both macrophages and dendritic cells (C). Fold induction of SOCS3 expression was calculated using the 2^ΔCt method using HPRT as a reference gene. Macrophages and dendritic cells from wild-type and Tyk2^-/- transgenic mice were pre-treated with IL-10 and were stimulated with 100 ng/ml LPS and the inhibition of TNF-α expression was determined at 2 and 24 hours (D). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

A stable monomeric form of human IL-10 has impaired activity.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type mice were tested for their response to human IL-10 and a stable monomeric form of IL-10 (IL-10m). Cells pre-treated with IL-10 were stimulated with 100 ng/ml LPS and TNF-α expression was determined to asses anti-inflammatory properties of IL-10 in macrophages (A) and dendritic cells (B) (<i>n</i> = 3, error bars indicate standard error). Mast cells were cultured for 48 hours in the presence of IL-10, where after cell viability was determined (C) (<i>n</i> = 3, error bars indicate standard error). <i>P</i><0.01 at all concentrations in all three assays.</p

IL-10R2 mediated signaling via Tyk2 plays a limited role in IL-10 activity.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells from wild-type, IL-10R1^-/-, IL-10R2^-/- and Tyk2^-/- mice were tested for their response to IL-10. Macrophages and dendritic cells from wild-type and IL-10R^-/- mice were pre-treated with IL-10 and subsequently stimulated with 100 ng/ml LPS. The percentage of inhibition of TNF-α expression of macrophages and dendritic cells was determined after overnight incubation (A and B, respectively) (<i>n</i> = 3, error bars indicate standard error). Similarly, macrophages and dendritic cells from Tyk2^-/- mice were tested for their response to IL-10 (D and E, respectively) (<i>n</i> = 4, error bars indicate standard error). Mast cells from wild-type and transgenic mice were cultured for 48 hours in the presence of IL-10 and cell viability was determined (C and F) (<i>n</i> = 3 for IL-10R^-/- mice and <i>n</i> = 4 for Tyk2^-/- mice, error bars indicate standard error). Asterisk(s) indicate significant differences as determined by a Welch’s <i>t</i>-test (*<i>P</i><0.05; **<i>P</i><0.01).</p

The differential response of bone marrow-derived cells to IL-10.

<p>Bone marrow-derived macrophages, dendritic cells and mast cells were analysed by flow cytometry for the expression of cellular markers CD11b & F4/80 (A), CD11c & MHC-II (D) and FcεRI & c-kit (G), respectively. Bone marrow-derived cells were tested for their response to IL-10 by measuring suppression of TNF-α expression by macrophages (B) and dendritic cells (E) or proliferative/anti-apoptotic ability in mast cells (H) (<i>n</i> = 4, error bars indicate standard error). Dose dependent tyrosine phosphorylation of STAT transcription factors by IL-10 (0, 1, 10 or 100 ng/ml) was analysed by western blot in macrophages (C), dendritic cells (F) and mast cells (I).</p

Analysis of the effect of <i>N</i>-glycosylation at Asn29 on granulation.

<p>Glycosylation of IL-10 plays a role in preventing granulation. (A/B) Whole mount confocal microscopy output of leaves expressing GFP fused C-terminally to human (h) and mouse (m) IL-10 including native signal peptide (SP) and with introduced (S29N) or removed (N29S) glycosylation site, respectively. (C/D) Western blot analysis under reducing conditions of plant produced (p) hIL-10 and mIL-10 with and without glycosylation site. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 and mIL-10 were used. A molecular weight marker is indicated in kDa. (E/F) Yield of hIL-10 and mIL-10 with and without glycosylation site in crude extracts 2 to 5 days post infiltration (dpi) as determined by ELISA (<i>n</i> = 4, error bars indicate standard error).</p

Analysis of expression of a stable monomeric form of human IL-10.

<p>A stable monomeric form of human IL-10 (hIL-10^mono) does not granulate and yield increases 30-fold. (A) Three cartoons illustrating the human IL-10 (I) dimer, (II) monomer and (III) stable monomer structure, as well as a schematic representation of the human (h) IL-10 alpha helices A–F. Helices are represented by ovals, whereby a fragment of the amino acid sequence and the location of insertion of the small GS-linker is indicated. (B) Whole mount confocal microscopy output of GFP fused C-terminally to hIL-10^mono including native signal peptide (SP). (C) Western blot analysis under non-reducing conditions of plant produced hIL-10 and hIL-10^mono. As controls, empty vector (EV) and 50 ng recombinant (r) <i>E. coli</i> produced hL-10 were used. A molecular weight marker is indicated in kDa. (D) Yield of hIL-10 and hIL-10^mono in crude extracts 2 to 5 days post infiltration as determined by ELISA (<i>n</i> = 3, error bars indicate standard error). Average yield of hIL-10^mono was significantly higher compared to hIL-10.</p

Expression data of human and mouse IL-10 in transiently transformed <i>Nicotiana benthamiana</i> leaves.

<p>Use of the thk-tag gives an increasing boost in yield for both human and mouse IL-10 from 2 days post infiltration (dpi). Strikingly, mouse IL-10 yield was significantly higher compared to human IL-10, regardless of ER-retention. Differences in yield could not be explained by differences in mRNA transcript levels. (A) Schematic representation of expression cassettes and vector used. Expressed genes include the native coding sequence of the human (h) or mouse (m) IL-10 gene including signal peptide for secretion (SP) with or without a 3′ tag coding for a thrombin cleavage site, a 6xHis-tag and the ER retention sequence KDEL (thk). All expression cassettes include the 35S promoter of the Cauliflower mosaic virus with duplicated enhancer (d35S), 5′ leader sequence of the Alfalfa mosaic virus RNA 4 (AlMV) and <i>Agrobacterium tumefaciens</i> nopaline synthase transcription terminator (Tnos). (B) Relative transcript levels of IL-10 versus actin as determined by Q-PCR on 2 and 3 dpi (<i>n</i> = 3, error bars indicate standard error). (C/D) Human and mouse IL-10 yield in crude extracts (1 to 6 dpi) in µg per mg total soluble protein (TSP) as determined by ELISA (<i>n</i> = 3, error bars indicate standard error).</p

Biological activity of human and mouse IL-10 variants on human and mouse macrophages.

<p>Plant produced (p) and recombinant (r) <i>E. coli</i> produced human (h) or mouse (m) IL-10 were calibrated to contain the same amount of IL-10 as well as total soluble protein by using the empty vector control. Human (THP-1) and mouse (RAW264.7) macrophages were then pretreated with 10 ng/ml hIL-10 or mIL-10 for 20 min and subsequently stimulated with 1 µg/ml <i>E. coli</i> lipopolysaccharide. Tumor Necrosis Factor-alpha (TNF-α) expression was determined by ELISA and IL-10 activity is indicated as the percentage of inhibition of TNF-α expression as compared to the empty vector control (<i>n</i> = 3, error bars indicate standard error).</p