IFNα-induced CXCL11 and CCL5 expression involves STAT2 and IRF9, but not STAT1.

<p>Cultured human keratinocytes were transfected with siRNA directed against STAT2 (siSTAT2), STAT1 (siSTAT1), IRF9 (siIRF9), or control siRNA (siCon) before IFNα (1000 U/ml) stimulation for 24 hours. (A) Protein extracts were isolated from the cells and the protein level of STAT2, STAT1, and IRF9 examined by western blotting. β-actin was used as a loading control. One representative gel out of three is shown. (B, C) The protein level of (B) CXCL11 and (C) CCL5 in the cell culture medium was analyzed by ELISA (n = 3). Results are expressed as mean ± standard deviation. *<i>P</i> < 0.05.</p

Increased mRNA expression of <i>CXCL11</i> and <i>CCL5</i> in lesional psoriatic skin.

<p>Total RNA was isolated from biopsies obtained from normal healthy volunteers as well as lesional and nonlesional psoriatic skin. mRNA expression of (A) <i>CXCL11</i> and (B) <i>CCL5</i> was analyzed by qPCR. <i>RPLP0</i> mRNA expression was used for normalization. Scatterplot shows the result from 6 healthy volunteers and 16 psoriatic patients. *<i>P</i> < 0.01.</p

STAT2-mediated regulation of CXCL11 and CCL5 expression.

<p>(A) Cultured human keratinocytes were stimulated with IFNα (1000 U/ml) for the indicated time points, and the phosphorylation level of STAT2 analyzed by western blotting (n = 3). (B) Keratinocytes were transfected with STAT2 siRNA (siSTAT2), control siRNA (siCon) or transfection reagent alone (Mock) before being stimulated with IFNα (1000 U/ml) for 24 hours. Protein extracts were isolated from the cells and STAT2 and P-STAT2 expression analyzed by western blotting (n = 3). β-actin was used as a loading control. (C) Human keratinocytes were transfected with STAT2 siRNA (siSTAT2) or control siRNA (siCon) before stimulation with IFNα (1000 U/ml) for 24 hours. Then the cell culture medium was screened for 102 proinflammatory cytokines and chemokines using a proteome profile array. Numbers on the membrane marks the following targets: 1 (CXCL11), 2 (IL-1ra), 3 (IL-1α), 4 (CCL5), 5 (MCP-1), 6 (IL-8), 7 (CXCL1) and 8 (ENA-78). (D, E) Human keratinocytes were stimulated as in (C) and the expression level of CXCL11 and CCL5 analyzed by ELISA (n = 3). Results are expressed as mean ± standard deviation. *<i>P</i> < 0.05.</p

STAT2 expression is elevated in psoriatic skin.

<p>(A) <i>STAT2</i> mRNA expression was examined by qPCR in biopsies obtained from normal healthy volunteers as well as from paired punch biopsies obtained from nonlesional (NLS) and lesional (LS) skin from patients suffering from psoriasis or atopic dermatitis (AD). The mRNA expression of <i>RPLP0</i> was used for normalization. Biopsies from 6 healthy volunteers, 16 psoriatic patients and 6 atopic dermatitis patients were examined. The results are presented as dot plots with the horizontal line expressing the mean value. All measurements were performed in triplicates. (B) Whole cell protein extracts were prepared from paired keratome biopsies taken from nonlesional and lesional psoriatic skin from five psoriatic patients. Phosphorylated STAT2 as well as total STAT2 protein was analyzed by western blotting. Equal protein loading was assessed by detecting the protein level of β-actin. Protein extract from keratinocytes stimulated with IFNα for 1 hour was included as a positive control (PC). MW; molecular weight marker. Densitometic analysis of the band intensity was carried out and values were normalized to β-actin. *<i>P</i> < 0.01. (C-E) Immunofluorescence analysis was performed on paraffin-embedded punch biopsies from (C) normal skin as well as (D) nonlesional and (E) lesional psoriatic skin. Nuclear staining was performed using 4’, 6-diamidine-2’-phenylindole dihydrochloride (DAPI). Green color (Alexa Fluor 488) represents STAT2 protein. Three sets of biopsies from three different patients were investigated. Arrows show nuclear staining. Scale bar = 100 μm.</p

The expression of <i>CXCL11</i> and <i>CCL5</i> is positively correlated with IFNγ expression in lesional psoriatic skin.

<p>(A) Chemotaxis assays were performed using a 24-well plate-based assay. PBMCs were isolated from blood taken from psoriatic patients and loaded on the upper chamber containing a 5-μm pore-size filter. Wells (lower chamber) were added vehicle or 10 nM recombinant CXCL11 or CCL20 and incubated for 20 hours. Cells migrating to the lower chamber were analyzed by flow cytometry (n = 9). Results are expressed as mean ± standard deviation. *<i>P</i> < 0.01. (B-E) RNA from punch biopsies taken from 16 psoriatic patients was isolated and the mRNA expression of <i>CXCL11</i>, <i>CCL5</i>, <i>IFNγ</i>, and <i>IL-17A</i> analyzed by qPCR. The correlation between the expression levels of these cytokines was analyzed as indicated in the figure.</p

IL-21R is expressed by vaginal epithelial cells in WT mice and IL-21R expression is increased by HSV-2 infection.

<p>8 weeks old C57BL/6 mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2 and vaginas were harvested on day 1-3 post infection (p.i.) (<b>A</b>) Quantitative RT-PCR analysis of mRNA encoding IL-21R in vaginal tissue in uninfected (u.i.) and HSV-2 infected WT mice on day 1-3 p.i. (<b>B-C</b>) IHC staining with IL-21R primary antibodies and HRP-linked goat anti-rabbit secondary antibodies on 2 µm sections of vaginal tissue in (<b>B</b>) u.i. and HSV-2 infected WT mice day 1 p.i. and in (<b>C</b>) HSV-2 infected WT and IL-21R KO mice day 1 p.i. as control. Scale bar  =  20 µm. (<b>A</b>) IL-21R is normalized to β-Actin in each sample. Horizontal bars indicate median. Each dot represents one animal. Data are representative of two individual experiments (<i>n</i>  =  10 mice per group respectively). ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Mice lacking the IL-21R are more susceptible to HSV-2 infection than WT mice.

<p>(<b>A-D</b>) 10 weeks old mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. Vaginal fluids were collected on day 1-3 p.i. for quantification of (<b>A</b>) vaginal viral load day 1-3 p.i. and (<b>B</b>) IFN-α/β protein levels in vaginal fluids day 2 p.i. The dashed line indicates the lower detection limit of the assay. (<b>C</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores for WT and IL-21R KO mice. (<b>D</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival, <i>p</i><0.001. (<b>A-B</b>) Horizontal bars indicate median. Each dot represents one animal. (<b>A-D</b>) Data are representative of two independent experiments (<i>n</i>  =  7 and 13 mice per group respectively, in A data are pooled). * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, # <i>p</i>  =  0.058 when using a Mann-Whitney test but when applying an unpaired Students t-test <i>p</i>  =  0.02. ns  =  not significant. (<b>E</b>) 20×10⁶ CD4⁺ splenocytes from Thy1.2 mice were adoptively transferred i.p. to Thy1.1 mice. Spleens were harvested the following day and splenocytes were analysed by flow cytometry for Thy1.2 and CD4. (<b>F-H</b>) 10 weeks old IL-21R KO mice were injected i.p. with 20×10⁶ splenocytes from 10 weeks old WT or IL-21R KO mice respectively. One day after the transfer, mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. (<b>F</b>) Vaginal fluids were collected on day 1-3 p.i. for quantification of vaginal viral load day 1-3 p.i. Horizontal bars indicate median. Each dot represents one animal. (<b>G</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores for IL-21R KO mice transferred with WT or IL-21R KO splenocytes. Differences in median disease score between the two groups did not reach statistical significance on any of the days. (<b>H</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival. ns  =  not significant. Data (<b>E-H</b>) represent one experiment.</p

Cytokine levels in vaginal fluids from HSV-2 infected IL-21R KO and WT mice.

<p>Vaginal fluids were collected on day 1-3 p.i. with 6.7×10⁴ PFUs HSV-2 intra-vaginally. (<b>A-F</b>) IL-6, IFN-γ, TNF-α, MCP-1 (CCL-2), KC (CXCL-1, murine homologue of IL-8) and IL-10 protein levels in vaginal fluids from HSV-2 infected IL-21R KO and WT mice, quantified by Luminex. Horizontal bars indicate median. Each dot represents one animal. Data are representative of two individual experiments (<i>n</i>  =  7 and 13, 10 weeks old mice, per group respectively). Statistical analysis has been performed on all data sets. Where differences between WT and IL-21R KO mice reached statistical significance it is indicated in the graph by * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. When differences did not reach statistical significance there is no indication in the graph.</p

Vaginal NK cell effector activity in IL-21R KO and WT mice day 2 p.i. with HSV-2.

<p>Vaginas were harvested on day 2 p.i. with 6.7×10⁴ PFUs HSV-2 intra-vaginally. (<b>A</b>) Example of intracellular IFN-γ and granzyme B staining in vaginal NK cells. (<b>B</b>) IFN-γ⁺ vaginal NK cells in % per mouse in IL-21R KO and WT mice. (<b>C</b>) Total number of IFN-γ⁺ vaginal NK cells per mouse in IL-21R KO and WT mice. (<b>D</b>) CD107a+b expression on FACS sorted viable CD45⁺ vaginal cells from WT mice, cultured in the presence or absence of MHC class 1 deficient YAC-1 cells, and gated on NK cell marker NK1.1. (<b>E</b>) CD107a+b⁺ NK cells in %, of FACS sorted viable CD45⁺ vaginal cells, cultured in the absence or presence of YAC-1 cells, and gated on NK1.1, in IL-21R KO and WT mice. (<b>A-C</b>) <i>n</i>  =  12 mice per group. (<b>D-E</b>) <i>n</i>  =  6 mice per group. (<b>B-C</b>) Horizontal bars indicate median. Each dot represents one animal. All mice were 8-9 weeks old. ns  =  not significant.</p

mIL-21 treatment has anti-viral effect in the innate immune response.

<p>8 weeks old WT mice were infected intra-vaginally with 6.7×10⁴ PFUs HSV-2. mIL-21 treated mice were inoculated intra-vaginally with 5 µg mIL-21 on day -1 to 3 p.i., and untreated mice received PBS. Vaginal fluids from WT mice infected with HSV-2 and untreated (-) or treated (+) with mIL-21, were collected on day 1-3 p.i. for quantification of (<b>A</b>) vaginal viral load day 1-3 p.i. and (<b>B</b>) IFN-α/β protein levels in vaginal fluids day 2 p.i. The dashed line indicates the lower detection limit of the assay. (<b>C</b>) After HSV-2 infection mice were scored daily for disease development. Data are shown as median disease scores in HSV-2 infected, untreated or mIL-21 treated WT mice. (<b>D</b>) When reaching score 4 (hind limb paralysis) mice were euthanized and recorded. Data are shown in a Kaplan Meier plot of survival, p<0.001. (<b>A-B</b>) Horizontal bars indicate median. Each dot represents one animal. (<b>A-D</b>) Data are representative of three individual experiments (<i>n</i>  =  10 mice per group). * <i>p</i><0.05, ** <i>p</i><0.01. ns  =  not significant.</p