C17 protein is monomeric.

<p>(A) Recombinant purified human C17-V5H8 was analyzed by SDS-PAGE under reducing (1) and non-reducing (2) loading conditions and subsequent Coomassi staining. A protein marker is shown in the right column (3). (B) Baseline drift-corrected A280 nm elution profile of size exclusion chromatography after loading affinity-purified human C17-V5H8. Retention times of marker proteins (in kDa) are indicated above.</p

Bone erosion and histological parameters of arthritis are reduced in mice over-expressing C17.

<p>One hind paw per animal was harvested at the time of euthanasia and was analyzed by micro-CT and subsequent histology. (A) Representative histology images from individual hind paws are shown either stained with H&E (top row) or stained with Safranin O (bottom row). Images were taken at 10× magnification. (B) Scoring of disease severity according to diverse histological parameters. (C) Representive micro-CT scans are shown. Red box highlights prominent joint erosion in paw of GFP treated mouse. Similar data were generated in at least three independent experiments with at least five animals per group.</p

Reduced expression of inflammatory markers and genes associated with joint destruction in paws of C17-treated animals.

<p>One hind paw per animal was harvested at the time of euthanasia and quantitative RT-PCR was performed to measure mRNA levels of cytokines associated with joint inflammation (A), and genes associated with joint remodeling and arthritic tissue destruction (B). (C) mRNA expression of selected genes from paws with severe swelling and matched clinical disease score 3. (D) C17 expression in paws from naïve animals and animals that received arthrogen with GFP control or C17 minicircle. Values from naïve animals are shown as filled triangles, from GFP mice as filled squares, from C17 mice as open circles, throughout the figure. Not significant: ns; p<0.05: *; p<0.01: **. Similar data were obtained in at least two independent experiments with five or more mice per group.</p

C17 mRNA and protein are expressed in cartilage-rich tissues and chondrocytes, respectively.

<p>(A) A variety of tissues were harvested and pooled from three C57BL/6 mice and then analyzed for C17 mRNA expression using qPCR (n.d., not detected). (B) Immunohistochemical staining for C17 on mouse sternal cartilage, using a mAb anti-C17, demonstrates protein expression by chondrocytes. Scale bars represent 0.1 mm.</p

Sustained over-expression of C17 does not result in an inflammatory response <i>in vivo</i>.

<p>Age-and sex-matched mice received a hydrodynamic injection of C17 or GFP (control) minicircle, or were kept naïve. (A) After five weeks, animals were euthanized and hepatic over-expression of C17 mRNA was verified. In addition, (B) hepatic expression of C17 protein was confirmed by IHC using an anti-C17 mAb (top row) or isotype control Ab (bottom row); arrowheads highlight areas of positive C17 staining in C17-transfected, but not naïve or GFP-transfected liver; images were taken at 20× magnification. (C) Systemic exposure to C17 was verified by measuring serum levels of tagged C17 protein. Data shown represent a typical result from at least two independent experiments with five or more animals per group.</p

C17 over-expression protects mice from collagen antibody-induced arthritis.

<p>Age-matched male B10.RIII mice received a hydrodynamic injection of GFP or C17 minicircle DNA in Ringer's solution. Three days later mice received a single dose of the 4-clone arthrogenic mAb cocktail (3 mg) to induce CAIA (day 0), mice were followed for eleven days and then euthanized. (A) Incidence of disease and (B) mean clinical disease scores ± SEM are shown. Thickness of both hind footpads from each animal was measured with a mechanical caliper at the time of euthanasia (C). Quantitative RT-PCR was performed on liver tissues to verify hepatic expression of C17 mRNA (D), and protein levels in the serum were measured by ELISA (E). Data shown here are representative of at least three independent experiments with at least five animals per group.</p

V5H8-peptide tag is not sufficient for protection from CAIA.

<p>Mice were injected with minicircle vector encoding GFP, V5H8-tagged IL-22BP, V5H8-tagged C17-V5H8,or untagged C17, on day −3. Artherogenic Ab cocktail was administered on day 0 and animals(n = 4–5/group) were monitored and scored daily relating to (A) disease incidence, and (B) clinical disease score. (C) Hind paw thickness was measured with a caliper on days 0 and 11, ***: p<0.001. (D) Presence of V5H8-tagged proteins in serum was verified on day 11; n.d., not detected.</p

C17 is related to the family of common gamma chain-dependent cytokines.

<p>ClustalW multiple protein sequence alignment with some manual adjustment. Sequences of human cytokines, structurally related to IL-2, were aligned. C17 is highlighted in purple. Blue sequence denotes structure-confirmed alpha-helical secondary structure elements of respective cytokine (IL-2, PDB 2B5I; IL-4, PDB 1ITM; IL-13, PDB 1IKO; IL-15, PDB 2PSM; IL-21, PDB 2QQP). Light blue background confirms amino acid residues providing contacts to the respective protein core, according to afore listed structure data. Grey background denotes amino acid residues with predicted protein core contacts. Symbols *, :, . denote different levels of amino acid similarity/identity. The arrow heads indicate two residues for predicted O-linked glycosylation that are conserved between human and mouse C17.</p

Human and mouse C17 are secreted proteins <i>in vitro</i>.

<p>HEK293 cells were transfected with eukaryotic expression vectors encoding C-terminally dual-tagged (V5H8 tags) human and mouse C17 protein. The tags were fused to the 3′ end of the full cDNA, retaining the natural N-terminal signal peptide and complete mature portion of C17. Supernatants and lysates were harvested and adjusted by volumes to permit direct comparison of protein quantities by Western blot. Proteins were separated by SDS-PAGE and visualized with an anti-poly His antibody. The same blot is shown twice with short (top) and long (bottom) exposure.</p

C17 over-expression does not modulate established arthritis.

<p>CAIA was induced in male B10.RIII mice with a single dose of 4-clone arthrogen on day 0, followed by hydrodynamic injection of GFP or C17-V5H8 minicircle on day 5 when mice had developed prominent paw inflammation. Mice were further monitored and euthanized at 17 days after initial disease induction. (A) The mean clinical disease scores including SEM are shown. (B) Histology gestalt scores were assigned to H&E-stained hind paws for GFP- and C17-treated animals. (C) Serum expression of C17 was verified. Data shown represent a typical result from at least two independent experiments with five or more animals per group.</p