Anti-inflammatory effect of a retrovirus-derived immunosuppressive peptide in mouse models

Two variants of the TPA-induced acute toxic contact eczema model were used to evaluate the anti-inflammatory potential of topical applied CKS-17 peptide. Initially, mice were pretreated for 3 days with peptide-containing cream followed by induction of ear inflammation by TPA. Figure 2A depicts the time-course. Evidently, mice treated with CKS-17 peptide have significantly reduced swelling of the ears upon inflammation induction. The direct comparison with a scrambled peptide in Figure 2B (day 1) points to a specific effect of CKS-17 dimer, which is significantly more potent than the scrambled peptide control to reduce ear inflammation (p < 0.001).

In the same model an experiment was performed in which inflammation was induced five days prior to treatment with peptide cream (Figure 3A). In this setup a monomer peptide vs a dimer was tested since the ISU-activity of the peptides are consistently correlated with di- or multimeric forms of the peptides[14, 19, 26]. This was confirmed in a direct comparison experiment, where CKS-17 dimer-containing cream significantly reduced ear inflammation in contrast to the monomeric peptide. Figure 3B clearly shows a significant reduction of inflammation two days after CKS-17 dimer treatment in contrast to the monomeric peptide (p = 0.003, Student’s t-test). Monomeric peptide was not different from the pure cream vehicle. Furthermore, in the same experiment we performed a direct comparison to a potent anti-inflammatory topical glucocorticoid based cream (Clobetasol propionate 0.05%). Clobetasol propionate treatment was very potent in this model and reduced inflammation close to the basal level with minimal inter-animal variation.

This model is representative of allergic contact dermatitis induced inflammation by sensitizing the mice 7 days prior to challenge. Three days before induction of inflammation the mice were treated with peptide-containing cream. Evidently, as in the TPA-induced eczema model treatment with CKS-17 containing cream lowered swelling of the ears (Figure 4A) following inflammation induction by oxazolone (day 0). However, the effect was less pronounced at day two post oxazolone challenge (p = 0.068 CKS-17 vs. scrambled, Student’s t-test) as shown in the box-plot in Figure 4B. In general, this contact dermatitis mouse model induces much less inflammation of the ears in comparison to the TPA mouse model. The results of a total of four independent experiments (3 eczema studies and 1 contact dermatitis) each with 6 mice pr treatment group are compiled in Figure 5. The figure depicts the relative reduction in ear thickness of the treatment groups (scrambled/monomer, CKS-17 or Clobetasol propionate) compared with TPA or Oxazolone alone. Across the experiments the reduction in the CKS-17 treatment group ranged from 20-43% with no effect of scrambled/monomer. Clobetasol propionate reduced the swelling of the ears by 80% in the single study where it was applied. The consistent reduction of treatment with CKS-17-containing cream independent of the model and scheme of application was significantly different from treatment with a scrambled peptide (p = 0.028, Mann Whitney test).

RNA from ear biopsies of the mice treated with TPA (mice depicted in Figure 2) was harvested and subjected to mRNA relative quantification of IL-1α, IL-6, IL-10, IL-17C, TNF-α and CXCL2. All expression levels were adjusted for input mRNA by GAPDH gene expression and the results normalized to levels for vehicle only treated mice. There was a clear and significant reduction of mRNA levels of IL-17C, TNF-α, IL-6 and CXCL2 in mice treated with CKS-17 containing cream (Figure 6, left column). When correlating the relative mRNA expression of these cytokines/chemokines to the ear thickness of individual animals, a statistically significant correlation was observed (Figure 6, right column). In turn, expression levels of neither IL-1α nor IL-10 were affected by treatment with CKS-17-containing cream compared to cream with scrambled peptide. Notably, plotting the expression levels of IL-1α and IL-10 against the ear thickness revealed no linear correlations where observed (Figure 6, right column). The results are summarized in Table 1.

Interestingly, treating mouse ears with cream containing CKS-17 peptide results in a potent blockage of TNF-α mRNA expression (mean index (range) 0.3(0.17-0.52)) to levels below the background in untreated animals in the vehicle control group (mean index (range) 1(0.49-1.51)).

CKS-17 peptide (LQNRRGLDLLFLKEGGLC) or scrambled CKS-17 (LGGEKLFLLDLGRRNQLC) both linked into dimers via the C-terminal cysteine were purchased as lyophilized powder from Anaspec (California, USA). CKS-17 monomer was purchased without the C-terminal disulphide linkage. All peptides were dissolved in sterile water.

BALB/cJ and C57BL/6 mice were purchased from Taconic Europe (Ry, Denmark). All mice used in this study were female and 6–8 weeks of age when experiments were initiated. The animals were kept in animal facilities that maintained a temperature of 19–25°C, and a 12-hour day/night cycle. They were given access to food and water ad libitum. All experiments were approved by the Committee for Animal Experiments in Denmark.

Two different dosings were pursued in this model. Firstly, C57BL/6 mice were treated once daily from day −3 until day 3 on the ears with cream (commercially available baby lotion; Natusan®, First Touch Protection cream) + 50% H₂O) alone, cream + scrambled peptide or cream + CKS-17 peptide. Before the first application of cream at day −3 the ear thickness was measured. At day 0 and day 2 (3 hours before treatment with cream) the mice were treated with phorbol 12-myristate 13-acetate (TPA)(Sigma-Aldrich, St Louis, MO, cat no. P8139) on the ears (2 μg/ear). TPA was dissolved in acetone. Before the first challenge of TPA the ear thickness was measured (day 0) and again post-challenge at the times indicated, using a Mitutyo digimatic indicator. In the second experiment, BALB/cJ were treated at day 0, 2, 5 and 7 with phorbol 12-myristate 13-acetate (TPA)(Sigma-Aldrich, St Louis, MO, cat no. P8139) on the ears (2 μg/ear). TPA was dissolved in acetone. Before the first challenge of TPA the ear thickness was measured (day 0) and again post-challenge at the times indicated, using a Mitutyo digimatic indicator. At day 5 and 7 the mice were treated one time with either cream alone, cream + CKS-17 monomér, cream + CKS-dimér or Clobetasolpropionat 0.05% cream formulation applied neat (GSK, UK). The mice were anaesthetised during the ear measurement.

4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one (oxazolone) was purchased from Sigma-Aldrich (cat no. E0753). Oxazolone was dissolved in acetone. At day −7 mice were sensitized with 1.5% oxazolone by application to the clipped abdomen (100 μl). Once daily from day −3 until day 1 the ears of the mice were treated with cream (commercial available baby lotion; Natusan®, First Touch Protection cream + 50% H₂O) alone, cream + scrambled peptide or cream + CKS-17 peptide. Before the first application of cream at day −3 the ear thickness was measured. At day 0 the mice were challenged with 0.5% oxazolone at the ears (20 μl per ear). This was done 3 hours prior to treatment with the different cream formulations. Before challenge (day 0), the ear thickness was measured and again post-challenge at the times indicated, using a Mitutyo digimatic indicator.

At termination of one study of acute toxic contact eczema punch biopsies (4 mm) from mice ears were transferred to 1 ml of −80°C cold RNAlater-ICE (Ambion inc., Austin, TX). Samples were kept at −80°C until 24 hours before RNA purification at which time they were transferred to −20°C. On RNA purification, biopsies were removed from RNAlater-ICE and transferred to 175 μl of SV RNA lysis buffer added β-mercaptoethanol (SV Total RNA Isolation System; Promega, Madison, WI) and homogenized. RNA purification was completed according to the manufacturer’s instructions (SV Total RNA Isolation System; Promega). RNA was stored until further use at −80°C.

For reverse transcription, Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, CA, U.S.A.) were used. For qPCR we used Platinum® qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA, U.S.A.) with primers and probes were Taqman 20× Assays-On-Demand (FAM-labeled MGB-probes) gene expression assay mix (Applied Biosystems). Each sample was loaded as triplets and analyzed on a Rotorgene-3000 real-time PCR machine (Corbett Research, Cambridge, U.K.). Relative gene expression levels were determined by using the relative standard curve method as outlined in User Bulletin 2 (ABI PRISM 7700 sequencing detection system, Applied Biosystems). Briefly, a standard curve for each gene was made of fourfold serial dilutions of total RNA from a punch biopsy from the ears of mice. The curve was then used to calculate relative amounts of target mRNA in the samples. As housekeeping GAPDH was used. Assay ID for the primers and probes used in this study were as follows: IL-17c (Mm00439619_m1); TNF-α (Mm00443258_m1); IL-1α (Mm00439620_m1); IL-6 (Mm00446190_m1); IL-10 (Mm00439616_m1); CXCL2 (Mm00436450_m1); GAPDH (Mm99999915_g1).

Comparisons of ear thickness and relative mRNA levels between treatment groups were performed using a student’s t-test. Correlations between ear thickness and mRNA levels were determined using spearman correlations. Lastly, the percentage effect on reduction in ear thickness between scrambled and CKS-17 treated groups across four independent mouse experiments were compared using a Mann–Whitney test. All p-values reported are two-tailed and we set the significance level at 5%.