The anaphase-promoting complex protein 5 (AnapC5) associates with A20 and inhibits IL-17-mediated signal transduction.

IL-17 is the founding member of a family of cytokines and receptors with unique structures and signaling properties. IL-17 is the signature cytokine of Th17 cells, a relatively new T cell population that promotes inflammation in settings of infection and autoimmunity. Despite advances in understanding Th17 cells, mechanisms of IL-17-mediated signal transduction are less well defined. IL-17 signaling requires contributions from two receptor subunits, IL-17RA and IL-17RC. Mutants of IL-17RC lacking the cytoplasmic domain are nonfunctional, indicating that IL-17RC provides essential but poorly understood signaling contributions to IL-17-mediated signaling. To better understand the role of IL-17RC in signaling, we performed a yeast 2-hybrid screen to identify novel proteins associated with the IL-17RC cytoplasmic tail. One of the most frequent candidates was the anaphase promoting complex protein 7 (APC7 or AnapC7), which interacted with both IL-17RC and IL-17RA. Knockdown of AnapC7 by siRNA silencing exerted no detectable impact on IL-17 signaling. However, AnapC5, which associates with AnapC7, was also able to bind IL-17RA and IL-17RC. Moreover, AnapC5 silencing enhanced IL-17-induced gene expression, suggesting an inhibitory activity. Strikingly, AnapC5 also associated with A20 (TNFAIP3), a recently-identified negative feedback regulator of IL-17 signal transduction. IL-17 signaling was not impacted by knockdown of Itch or TAXBP1, scaffolding proteins that mediate A20 inhibition in the TNFα and IL-1 signaling pathways. These data suggest a model in which AnapC5, rather than TAX1BP1 and Itch, is a novel adaptor and negative regulator of IL-17 signaling pathways

C/EBPβ^-/- mice exhibit increased susceptibility to oral candidiasis in the context of cortisone-induced immunosuppression.

<p><b>(A)</b> C65BL/6 mice (“WT”) were treated with the indicated doses of cortisone acetate at days -1, +1 and +2 relative to infection. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. SHAM (n = 3), No cortisone control (n = 5), 60mg/kg (n = 9), 120/112mg/kg (n = 8), and 225mg/kg (n = 8). Data are pooled from 2 independent experiments. Bars indicate geometric mean with 95% CI. P<0.05 by t-test with Mann-Whitney correction: * vs NO CORT, ≠ vs 60mg/kg, # vs 120/112mg/kg and π vs 225mg/kg. (<b>B</b>) The indicated mice were infected orally as described in panel A. Cortisone acetate was administered subcutaneously on days -1, +1 and +2 relative to infection. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/+ 60 mg/kg (n = 16), C/EBPβ^+/- 60mg/kg (n = 16), C/EBPβ^-/- 60mg/kg (n = 10) and C/EBPβ^+/+ 225mg/kg (n = 4). <i>P</i><0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. Data are pooled from two independent experiments. <b>C.</b> Representative tongue sections from the indicated mice were stained with H&E or Periodic-acid Schiff (PAS). Scale bar indicates 200 μM. White arrows indicate hyphae.</p

Susceptibility of C/EBPβ^-/- mice to OPC correlates with expression of BD3.

<p><b>(A)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from two independent experiments. <i>P</i><0.05 by student unpaired t-test. * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg. (<b>B)</b> mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection and analyzed as in panel A. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 2), C/EBPβ^+/- (n = 2), and C/EBPβ^-/- (n = 2). Data are from one experiment. * <i>P</i><0.05 by student unpaired t-test. vs C/EBPβ^+/+, ≠ vs C/EBPβ^+/- and π vs C/EBPβ^-/-. (<b>C)</b> OKF6/TERT2 human oral keratinocytes were treated with 200 ng/ml IL-17 plus 2ng/ml TNFα or with 2×10⁶ HK <i>C</i>. <i>albicans</i> for 24 h. Complementary DNA was prepared and subjected to qPCR analysis to detect <i>DEFB4A</i>. Data are normalized to expression of <i>GAPDH</i> and represent absolute levels. Data are representative of 2 independent experiments. *<i>P</i><0.05 compared to unstimulated OKF6/TERT2 cells.</p

The susceptibility of C/EBPβ^-/- mice to OPC does not correlate with expression of prototypical IL-17-regulated genes.

<p>mRNA from tongue was isolated from the indicated mice 5 days after oral <i>C</i>. <i>albicans</i> infection [C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ NO CORT (n = 3), C/EBPβ^+/+ 60mg/kg (n = 5), C/EBPβ^+/- 60mg/kg (n = 6), and C/EBPβ^-/- 60mg/kg (n = 5)]. Complementary DNA was prepared and subjected to qPCR analysis to detect the indicated genes. Results are presented as fold induction over SHAM treated mice and normalized to expression of <i>Gapdh</i>. Data are pooled from 2 independent experiments. <i>P</i><0.05 by student unpaired t-test: * vs C/EBPβ^+/+ NO CORT, ≠ vs C/EBPβ^+/+ 60mg/kg, # vs C/EBPβ^+/- 60mg/kg and π vs C/EBPβ^-/- 60mg/kg.</p

(Simpson-Abelson <i>et al</i>.).

<p>Transcript expression levels of the indicated genes in tongues isolated from the indicated mice 5 days after induction of oropharyngeal candidiasis. Increase over Sham-infected mice is indicated as follows:</p><p>*2-4-fold,</p><p>** 5–10 fold,</p><p>*** 10–50 fold,</p><p>**** > 100-fold</p><p>(Simpson-Abelson <i>et al</i>.).</p

C/EBPβ^-/- mice exhibit increased susceptibility to systemic candidiasis but are resistant to oral candidiasis.

<p>(<b>A</b>) The indicated mice (n = 3 per group) were injected with <i>C</i>. <i>albicans</i> in the lateral tail vein. Time to sacrifice is indicated (days). *<i>P</i><0.05 versus (vs) C/EBPβ^+/- mice using Log-rank (Mantel-Cox). (<b>B</b>) The indicated mice were infected sublingually with <i>C</i>. <i>albicans</i> for 75 mins. After 5 d, fungal loads in tongue were assessed by CFU enumeration of tongue tissue homogenates. Bars indicate geometric mean with 95% CI. C/EBPβ^+/+ SHAM (n = 3), C/EBPβ^+/+ (n = 5), C/EBPβ^+/- (n = 9), C/EBPβ^-/- (n = 3), C/EBPβ^+/+ plus 225mg/kg cortisone (n = 3); cortisone acetate was administered by subcutaneous injection on days -1, +1 and +2 relative to infection. <i>P</i><0.05 by t-test with Mann-Whitney correction: * vs C/EBPβ^+/+ WT, ≠ vs C/EBPβ^+/-, # vs C/EBPβ^-/-, π vs C/EBPβ^+/+ 225mg/kg. <b>C</b>. Weights of mice were assessed daily and graphed as percent of starting weight. * vs C/EBPβ^+/+ WT. <i>P</i><0.05 by t-test with Mann-Whitney correction. Experiment was performed once.</p

Neither Rrad nor CFD mediate IL-17 signal transduction. A. Rrad-deficient fibroblasts mediate normal IL-17 induction of IL-6 secretion.

<p>Multiple Rrad^−/− cell lines (derived from adult tail biopsies of Rrad^−/− mice) or ST2 stromal cell lines were treated with IL-17 (200 /ng/ml), IL-17F (200/ ng/ml) or suboptimal TNFα (2 ng/ml) for 24 h. IL-6 was measured in culture supernatants in triplicate by ELISA. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. B. Rrad-deficient fibroblasts mediate normal IL-17 induction of target gene expression. A representative Rrad^−/− cell line or IL-17RC^−/− fibroblasts were treated with IL-17 or TNFα as outlined in panel A for 24 h. Expression of the indicated genes was assessed by qPCR in triplicate. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls of each cell line. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. C. Complement deficient cell lines mediate normal IL-17 signaling. Fibroblast cell lines from CFD^−/−, C3^−/− or IL-17RC^−/− mice were treated with IL-17 and TNFα as described in panel A, and IL-6 concentrations in culture supernatants were assessed by ELISA. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. ^¶concentration values above standard curve for ELISA detection. All data are representative of at least two independent experiments.</p

Schematic model of AnapC5 and AnapC7 in IL-17R signaling.

<p>The data in this paper support a model in which AnapC5 serves an adaptor or scaffold protein to facilitate A20 recruitment to the CBAD domain of IL-17RA. Although AnapC7 binds to both IL-17RA and IL-17RC, its functional role is still unclear.</p

AnapC5 binds to the IL-17 receptor and restricts IL-17-mediated signal transduction. A. AnapC5 associates with IL-17RA, IL-17RC and AnapC7.

<p>HEK293T cells were transfected with AnapC5 (tagged with HA) together with IL-17RA, IL-17RC or AnapC7 (tagged with Myc), as indicated. Lysates were subjected to IP with anti-Myc Abs and immunoblotted with anti-HA or anti-Myc. Arrows indicate identity of each Myc-tagged protein. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. B. AnapC5 associates with IL-17RA through the inhibitory CBAD domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RA deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-Myc. Migration of protein size markers is indicated. C. AnapC5 associates with IL-17RC in the SEFIR domain. HEK293T cells were transfected with AnapC5 and the indicated IL-17RC deletion constructs. Lysates were subjected to co-IP with anti-Myc and blotted for HA or Myc. Whole cell lysates (WCL) were verified for AnapC7 by staining with anti-HA. Migration of protein size markers is indicated. D. Knockdown of AnapC5 enhances IL-17 signaling. ST2 cells were transfected with the indicated siRNAs, stimulated with IL-17 for 24 h, and IL-6 in culture supernatants assessed by ELISA. <i>n.s.,</i> not significant. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. E. Efficient knockdown of AnapC5 and AnapC7. mRNA from the samples in panel D were assessed for AnapC5 and AnapC7 expression by qPCR. *<i>p</i><0.05 by ANOVA and post-hoc Tukey’s test compared to unstimulated controls. ^‡<i>p</i><0.05 by Chi Square comparing experimental replicates. Data are representative of at least 2 independent experiments.</p